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Team:Heidelberg/Tyrocidine
From 2013.igem.org
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| - | Our strain <i>Brevibacillus Parabrevis</i> arrived from the Marahiel-lab. As preparation for the module shuffling experiments the strain was validated with test primers by colony PCR. The screening was positive, so we designed our Gibson compatible | + | Our strain <i>Brevibacillus Parabrevis</i> arrived from the Marahiel-lab. As preparation for the module shuffling experiments the strain was validated with test primers by colony PCR. The screening was positive, so we designed our Gibson compatible primers for amplification. </p> |
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<h1>Week 13</h1> | <h1>Week 13</h1> | ||
| - | <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We started with the amplification of fragments for the module shuffling with Gibson Primers. Most fragments were amplified in the first attempt, however others needed protocol optimization. At the end of the week 12 out of 15 fragments were successfully amplified. The next step to take is to amplify the remaining fragments and start with the Gibson Assembly. </p> | + | <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">We started with the amplification of fragments for the module shuffling with Gibson Primers. Most fragments were amplified in the first attempt, however others needed protocol optimization. At the end of the week 12 out of 15 fragments were successfully amplified. The next step to take, is to amplify the remaining fragments and start with the Gibson Assembly. </p> |
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<div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0"> | <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0"> | ||
<h1>Week 14</h1> | <h1>Week 14</h1> | ||
| - | <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After DNA concentration measurements, some of the previously amplified fragments had to be reamplified. The PCR protocols were optimized and the missing fragments were successfully obtained. We amplified all fragments with the necessary concentration to start | + | <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">After DNA concentration measurements, some of the previously amplified fragments had to be reamplified. The PCR protocols were optimized and the missing fragments were successfully obtained. We amplified all fragments with the necessary concentration to start with the Gibson assembly. However we noticed that we mixed up fragments during the assembly of Tripeptide-I-NRPS (pIK04)and resumed this experiment in the next week. </p> |
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<div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0"> | <div class="carousel-caption scrollContent2" data-spy="scroll" data-target="#navbarExample" data-offset="0"> | ||
<h1>Week 16</h1> | <h1>Week 16</h1> | ||
| - | <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;">Module Shuffling | + | <p style="font-size:10pt; text-align:justify; position:relative; margin-left:6%;"><b>Module Shuffling</b> |
Sequencing proved the successful assembly of the Tripeptide-I-NRPS (pIK04). All other NRPs had to be reconstructed through a new Gibson Assembly. For this purpose, we optimized the Gibson Assembly master mix, assuming that backbone religations were one of the main reasons for the failed assembly. We calculated a lower backbone-to-insert ratio and added fragment 12 in excess to the Tetrapeptide-I-fragment-mix. The assembled plasmids were transformed into electrocompetent DH10β and plated onto Cm-Lb. Afterwards white colonies were picked and grown overnight in 2x YT medium (Cm). Their mini-preps were analyzed with restriction digest. Two samples of each the Dipeptide (pIK03) and the Tetrapeptide-I (pPW01) plasmids were sent to sequencing. There was no positive restriction digest, neither for the Tetrapeptide-I-NRPS (pPW01) nor for Tetrapeptide-II-NRPS (pPW02). We decided not to reassemble the Tetrapeptide-II-construct (pPW02) and did not continue working on this NRP. Hence only colonies on the Tetrapeptide-I-construct (pPW01) plates were picked. | Sequencing proved the successful assembly of the Tripeptide-I-NRPS (pIK04). All other NRPs had to be reconstructed through a new Gibson Assembly. For this purpose, we optimized the Gibson Assembly master mix, assuming that backbone religations were one of the main reasons for the failed assembly. We calculated a lower backbone-to-insert ratio and added fragment 12 in excess to the Tetrapeptide-I-fragment-mix. The assembled plasmids were transformed into electrocompetent DH10β and plated onto Cm-Lb. Afterwards white colonies were picked and grown overnight in 2x YT medium (Cm). Their mini-preps were analyzed with restriction digest. Two samples of each the Dipeptide (pIK03) and the Tetrapeptide-I (pPW01) plasmids were sent to sequencing. There was no positive restriction digest, neither for the Tetrapeptide-I-NRPS (pPW01) nor for Tetrapeptide-II-NRPS (pPW02). We decided not to reassemble the Tetrapeptide-II-construct (pPW02) and did not continue working on this NRP. Hence only colonies on the Tetrapeptide-I-construct (pPW01) plates were picked. | ||
The positive Dipeptide- (pIK03), Tripeptide-I (pIK04) and Tetrapeptide-I (pPW01) NRPS constructs were chemically transformed into BAP I cells and plated onto Cm-LB. Furthermore colonies were picked and validated through restriction digest. Here only pIK03 (Dipeptide) and pIK04 (Tripeptide-I) were positive. The Tetrapeptide-I-NRPS (pPW01) was chemically transformed a second time into BAP I. | The positive Dipeptide- (pIK03), Tripeptide-I (pIK04) and Tetrapeptide-I (pPW01) NRPS constructs were chemically transformed into BAP I cells and plated onto Cm-LB. Furthermore colonies were picked and validated through restriction digest. Here only pIK03 (Dipeptide) and pIK04 (Tripeptide-I) were positive. The Tetrapeptide-I-NRPS (pPW01) was chemically transformed a second time into BAP I. | ||
| - | NRPS-Library | + | <b>NRPS-Library</b> |
For the standardization of the modules from the Tyrocidine cluster used in the shuffling we started amplification with the previously ordered Primers for Modules tycAdCom, tycB1dCom, tycC5, tycC6 introducing the RFC10 prefix and suffix. However, errors in the reverse Primers for tycC5 and tycC6 were noticed and re-ordered. Still the amplification of modules tycAdCom and tycB1dCom was started, but could not be amplified in the necessary concentrations. | For the standardization of the modules from the Tyrocidine cluster used in the shuffling we started amplification with the previously ordered Primers for Modules tycAdCom, tycB1dCom, tycC5, tycC6 introducing the RFC10 prefix and suffix. However, errors in the reverse Primers for tycC5 and tycC6 were noticed and re-ordered. Still the amplification of modules tycAdCom and tycB1dCom was started, but could not be amplified in the necessary concentrations. | ||
| - | Interspecies Module Shuffling | + | <b>Interspecies Module Shuffling</b> |
The primers for the interspecies NRPS fusion of different Tyrocidine modules with the Indigoidine synthetase indC were designed and ordered. </p> | The primers for the interspecies NRPS fusion of different Tyrocidine modules with the Indigoidine synthetase indC were designed and ordered. </p> | ||
<p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p> | <p><a class="btn btn-large btn-primary" href="#">Browse gallery</a></p> | ||
Revision as of 16:36, 2 October 2013
Project
Notebook
Human Practice
Tyrocidine. Proving Modularity of NRPS by Shuffling Modules.
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Week 12
Our strain Brevibacillus Parabrevis arrived from the Marahiel-lab. As preparation for the module shuffling experiments the strain was validated with test primers by colony PCR. The screening was positive, so we designed our Gibson compatible primers for amplification.
Week 13
We started with the amplification of fragments for the module shuffling with Gibson Primers. Most fragments were amplified in the first attempt, however others needed protocol optimization. At the end of the week 12 out of 15 fragments were successfully amplified. The next step to take, is to amplify the remaining fragments and start with the Gibson Assembly.
Week 14
After DNA concentration measurements, some of the previously amplified fragments had to be reamplified. The PCR protocols were optimized and the missing fragments were successfully obtained. We amplified all fragments with the necessary concentration to start with the Gibson assembly. However we noticed that we mixed up fragments during the assembly of Tripeptide-I-NRPS (pIK04)and resumed this experiment in the next week.
Week 15
The DNA concentration for all obtained fragments were measured and used for the calculation of the Gibson Assembly master mix. All five NRPS constructs producing the Dipeptide, Tripeptides I & II and Tetrapeptides I & II were assembled into a pSB1C3 backbone, electroporated into DH10β and spread on Chloramphenicol-LB plates. Several white colonies were picked and grown in 2x YT medium. Their mini-preps where validated through restriction digest. Positive samples were sent to sequencing. The results arrived in week 16. For the detection of the short synthetic NRPs we decided on using Mass Spectrometry (MS) and contacted a MS facility on campus.
Week 16
Module Shuffling Sequencing proved the successful assembly of the Tripeptide-I-NRPS (pIK04). All other NRPs had to be reconstructed through a new Gibson Assembly. For this purpose, we optimized the Gibson Assembly master mix, assuming that backbone religations were one of the main reasons for the failed assembly. We calculated a lower backbone-to-insert ratio and added fragment 12 in excess to the Tetrapeptide-I-fragment-mix. The assembled plasmids were transformed into electrocompetent DH10β and plated onto Cm-Lb. Afterwards white colonies were picked and grown overnight in 2x YT medium (Cm). Their mini-preps were analyzed with restriction digest. Two samples of each the Dipeptide (pIK03) and the Tetrapeptide-I (pPW01) plasmids were sent to sequencing. There was no positive restriction digest, neither for the Tetrapeptide-I-NRPS (pPW01) nor for Tetrapeptide-II-NRPS (pPW02). We decided not to reassemble the Tetrapeptide-II-construct (pPW02) and did not continue working on this NRP. Hence only colonies on the Tetrapeptide-I-construct (pPW01) plates were picked. The positive Dipeptide- (pIK03), Tripeptide-I (pIK04) and Tetrapeptide-I (pPW01) NRPS constructs were chemically transformed into BAP I cells and plated onto Cm-LB. Furthermore colonies were picked and validated through restriction digest. Here only pIK03 (Dipeptide) and pIK04 (Tripeptide-I) were positive. The Tetrapeptide-I-NRPS (pPW01) was chemically transformed a second time into BAP I. NRPS-Library For the standardization of the modules from the Tyrocidine cluster used in the shuffling we started amplification with the previously ordered Primers for Modules tycAdCom, tycB1dCom, tycC5, tycC6 introducing the RFC10 prefix and suffix. However, errors in the reverse Primers for tycC5 and tycC6 were noticed and re-ordered. Still the amplification of modules tycAdCom and tycB1dCom was started, but could not be amplified in the necessary concentrations. Interspecies Module Shuffling The primers for the interspecies NRPS fusion of different Tyrocidine modules with the Indigoidine synthetase indC were designed and ordered.
Week 17
Validation and characterization of BAP I cells transformed with the Dipeptide- (pIK03), Tripeptide-I- (pIK04) and Tetrapeptide-I-synthetase (pPW01) constructs through restriction digest. The pIK03 and pIK04 were positive and sent to sequencing. The restriction digest for pPW01 was negative. I a new strategy it was therefore co-transformed with a plasmid (pRB15) carrying the PPTase sfp into electrocompetent DH10β. Again the restriction digest was negative and we decided to abandon this construct assuming it was too big for simple transformation. For the Mass Spectrometry (MS) we needed to find a clone strongly expressing the NRPS. Therefore we performed a SDS-PAGE, which failed due to systematic errors. This was resumed the following week. The RFC10 standardization of the modules still showed to be difficult due to the total length of prefix/suffix + binding sequence (resulting in high annealing temperatures). To be efficient, we focused on the optimization of the amplification protocol for tycAdCom. We succeeded and adapted our protocols in the same manner for the other modules. Again the amplification for tycB1 and tycC5 failed, however we were able to amplify the module tycC6. After many attempts we redesigned the primers for tycB1dCom (fw and rv) and tycC5 (fw). The experiments concerning the synthesis of the interspecies fusion of a Tyrocidine module and the Indigoigine synthetase were started with the amplification of the fragments needed for introduction of the three constructs (coding for Phe-/Asn-/Val-Ind-Synthetases) into their backbone pSB1C3 by Gibson Assembly. All of those fragments were obtained in the necessary concentrations after optimization of the protocols.
Week 18
We started the week with a SDS-PAGE analyzing the expression capacity of several clones for the Dipeptide- and Tripeptide-I-NRPS. It showed a positive band for the Dipeptide synthetase (212 kDa) and an inconclusive band for the Tripeptide-I synthetase (381 kDa), because the top band of our ladder was at 260 kDa). Hence we interpreted this as a positive result for Tripeptide-I-synthetase as well. New primers for the tycB1dCom and tycC5 modules arrived and new protocols led to the successful amplification of the fragments. A restriction digest with EcoRI and PstI was executed to linearize the backbone pSB1C3 and to prepare the fragments for ligation. The Ligation was performed with T4 Ligase and then transformed into TOP10 cells. White clones where screened with a colony PCR (VF2 and VR primers) and one positive sample for each RFC10 compatible module-construct was sent to sequencing. Our fusion experiment was continued for all three constructs with the Gibson Assembly into pSB1C3. The mix was electroporated into DH10β and plated onto Cm-LB. The analytical restriction digest of picked colonies was positive for all constructs. Selected samples of each were proven to be positive by sequencing. Thereafter, the constructs were chemically transformed into our expression strain BAPI and spread onto Cm-LB plates. The colonies turned blue after less than two days, indicating the expression of Indigoidine. First a SDS-PAGE was executed showing that only the band for the Fusion NRPS producing Val-Ind was positive. For this clone purified Valine-Indigoidine from liquid culture was run next to native Indigoidine in a comparative TLC. Here it could be shown that the product from the Val-Ind-Synthetase runs slower than native Indigoidine. At this point it was already possible to assume that Indigoidine could be used to tag other NRP synthetase products by fusing it at the end of NRPS coding genes. To prove this assumption we designed a new strategy for the fusion of the indC to up to four Tyrocidine modules. For this plan new Gibson Assembly compatible primers were designed and ordered.
Week 19
Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.
Week 20
Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.
Week 21
Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.
Week 22
Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.
Week 23
Cras justo odio, dapibus ac facilisis in, egestas eget quam. Donec id elit non mi porta gravida at eget metus. Nullam id dolor id nibh ultricies vehicula ut id elit.
Methods:
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. .
06-07-2013
Amplification from FS_02 to FS_03; 5.3 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 1 |
| FS_03: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 6/7 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 min | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification worked with 5% DMSO
- Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices
03-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
| µL 1st PCR | what | µL 2nd PCR |
|---|---|---|
| 1 | D. acidovorans DSM-39 | 1 |
| 2.5 | FS_02 (1/10) | 2.5 |
| 2.5 | FS_05 (1/10) | 2.5 |
| 25 | Phusion Master Mix | 25 |
| - | DMSO | 2.5 |
| 19 | dd H2O | 19 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- sample has to be purified before further use since contamination with propanol was present
04-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
| what | µL 2nd PCR |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02 (1/10) | 2.5 |
| FS_05 (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit resulting in a final concentratio of 10ng/µL
- PCR will be repeated and gel slices purified with QIAX II Gel Extraction Kit, which is specifically designed to deliver higher yields when purifying fragments with sizes over 10 kbp
05-08-2013
Amplification from FS_02 to FS_07; 16.4 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 1 |
| FS_07: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 6 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 5min 40s | |
| 24 | 98 | 1 |
| 60 | 5 | |
| 72 | 5min 40s | |
| 1 | 72 | 17 min |
| 1 | 4 | inf |
Results:
- amplification of DelAG did not work
- repeat PCR with higher annealing temperature to increase specifity
06-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 1 |
| FS_07: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 6/7 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelEG worked but another unexpected product was amplified as well
- band was cut out avoiding contamination with other product and DNA purified using QIAquick Gel Extraction Kit
03-07-2013
Amplification from FS_06 to FS_09; 8.5 kb
- Reaction
| µl 1st PCR | what | µl 2nd PCR |
|---|---|---|
| 1 | D. acidovorans DSM-39 | 1 |
| 2.5 | FS_06 (1/10) | 2.5 |
| 2.5 | FS_09 (1/10) | 2.5 |
| 25 | Phusion Master Mix | 25 |
| - | DMSO | 2.5 |
| 19 | dd H2O | 19 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelFG did not work
- PCR will be repeated with a lower annealing temperature of 68°C (touchdown)
Amplification from FS_06 to FS_09; 8.5 kb
- Reaction
| what | µl 2nd PCR |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_06 (1/10) | 2.5 |
| FS_09 (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelFG did not work, many different side product occured, PCR will be repeated with other primers
Amplification from FS_06 to FS_11; 11.6 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_06: (1/10) | 1 |
| FS_11: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
05-07-2013
Amplification from FS_06 to FS_07; 5.2 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_06: (1/10) | 1 |
| FS_07: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 14 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 3:20 min | |
| 16 | 98 | 1 |
| 60 | 5 | |
| 72 | 3:20 min | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
05-07-2013
Amplification from FS_08 to FS_11; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 1 |
| FS_11: (1/10) | 1 |
| Phusion Master Mix | 10 |
| dd H2O | 6 |
| DMSO | 1 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 14 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 3:20 min | |
| 16 | 98 | 1 |
| 60 | 5 | |
| 72 | 3:20 min | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
Results:
- A weak band was visible, but it was on the wrong height.
- Accidently the band was cut anyway.
- Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.
07-07-2013
Amplification from FS_08 to FS_11; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2.5 |
| FS_11: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| dd H2O | 19 |
| DMSO | 2.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 min | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- There was no band visible on the gel.
- Either the primers did not bind or the DNA still had to many secondary structures --> the consequence is to change the annealing temperature.
04-07-2013
Amplification from FS_12 to FS_13; 2.7 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_12: (1/10) | 1 |
| FS_13: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 40 | |
| 1 | 72 | 420 |
| 1 | 4 | inf |
Results:
- Amplification of DelOP did not work
- Amplification will be repeated to exclude that pipetting errors were the reason for the failure
04-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_14: (1/10) | 1 |
| FS_15: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 40 | |
| 1 | 72 | 7 min |
| 1 | 4 | inf |
Results:
- Amplification worked very well, we had a bright band on the right height.
- Band was cut out and DNA purified using QIAquick Gel Extration Kit.
06-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_14: (1/10) | 1 |
| FS_15: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 6/7 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 42 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 42 | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
05-07-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_16: (1/10) | 1 |
| FS_01: (1/10) | 1 |
| Phusion Master Mix | 10 |
| dd H2O | 6 |
| DMSO | 1 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 14 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 3:20 min | |
| 16 | 98 | 1 |
| 60 | 5 | |
| 72 | 3:20 min | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
Results:
- Amplification worked well and band was at expected height. Nevertheless the gel band did not run properly on the gel.
08-07-2013
Amplification from FS_02 to FS_03; 5.3 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2.5 |
| FS_03: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 19 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 min | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAE worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
12-07-2013
Amplification from FS_02 to FS_07; 16.4 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2 |
| FS_07: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 5:30 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 5:30 | |
| 1 | 72 | 15min |
| 1 | 12 | inf |
Results:
- only a smear occured, no specific product was amplified
- PCR will be repeated with a lower, constant annealing temperature
09-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 1 |
| FS_07: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions I
| Cycles-PCR | temperature [°C] | Time [s] |
|---|---|---|
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
- Conditions II
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 5 | 98 | 1 |
| 68 | 5 | |
| 72 | 3 min | |
| 25 | 98 | 1 |
| 72 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelEG did not work
11-07-2013
Amplification from FS_04 to FS_11s; 17.5 kb
.png)
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 1 |
| FS_11_short: (1/10) | 1 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 5:30 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 5:30 | |
| 1 | 72 | 15min |
| 1 | 12 | inf |
Results:
- Amplification of DelEG did not work
- Experiment will be repeated with NEB Phusion II Polymerase as Phusion II is not provided as mastermix and therefore GC-buffer can be used
Amplification from FS_04 to FS_11; 17.5 kb; Phusion II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 1 |
| FS_11_short: (1/10) | 1 |
| Phusion II | 0.2 |
| DNTP | 0.4 |
| Buffer | 4 |
| DMSO | 0.6 |
| dd H2O | 11.8 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 30 |
| 12 | 98 | 5 |
| 68 ↓ 0.5 | 30 | |
| 72 | 8:10 | |
| 18 | 98 | 5 |
| 66 | 30 | |
| 72 | 8:10 | |
| 1 | 72 | 15min |
| 1 | 17 | inf |
Results:
- Amplification of DelEG did not work
- it seems not to be possible to amplify the desired 17 kbp fragent with the chosen primers and the given template, primercombination will be changed in further amplification attempts
14-07-2013
Amplification from FS_04 to FS_07; 11.1 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 2 |
| FS_07: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
Cycler incubation room right
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:00 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:00 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
- Conditions II
Cycler incubation room left
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 70 ↓ 0.5 | 5 | |
| 72 | 3:00 min | |
| 18 | 98 | 1 |
| 68 | 5 | |
| 72 | 3:00 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
Results:
- Amplification of DelEG worked with a touchdown PCR starting from 70°C annealing temperature
- band was cut out and DNA purified using QIAquick Gel Extraction Kit
- PCR will be repeated to increase the amount of DNA and gather the concentrations necessary for Gibson Assembly
09-07-2013
Amplification from FS_08 to FS_11_short; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08 (1/10) | 1 |
| FS_11_short (1/10) | 1 |
| Phusion Master Mix | 10 |
| dd H2O | 6 |
| DMSO | 1 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 2:30 | |
| 24 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 | |
| 1 | 72 | 10min |
| 1 | 4 | inf |
Results:
- A weak band was visible at the right height.
- Band was cut out and DNA purified using QIAquick Gel Extration Kit.
- Concentration after gel extration was too low
- Maybe increasing the temperature further will result in higher yield.
13-07-2013
Amplification from FS_10 to FS_11s; 3.3 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_10: (1/10) | 2 |
| FS_11_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 4 |
| DMSO | 1 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 70 | 5 | |
| 72 | 1:10 min | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
Results:
- No band was visible on the gel.
- The PCR conditions of the 09-07-2013 should be further optimized.
Amplification from FS_10 to FS_11(s); 3.3 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_10: (1/10) | 2 |
| FS_11 (short or long): (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 4 |
| DMSO | 1 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:10 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:10 min | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
Results:
- Bright bands were visible in both, the PCR with the short and the long primer.
- The PCR with the short primers worked better than the one with the long primer.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
- Concentration after gel extraction with primer FS_11short=6ng/µl in 18µl
- Concentration after gel extraction with primer FS_11long=4ng/µl in 18µl
11-07-2013
Amplification from FS_13s to FS_15s; 6.4 kb
_cut.png)
- Reaction of DelLP
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_13_short: (1/10) | 1 |
| FS_15_short: (1/10) | 1 |
| Phusion flash Master Mix | 10 |
| dd H2O | 6 |
| DMSO | 1 |
- Conditions of Del LP
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 2:10 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 2:10 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- There was a very weak band visible on the right height.
- The band was cut out anyway, as we wanted to use it as template for a Re-PCR
- The DNA was purified using QIAquick Gel Extration Kit.
12-07-2013
Re-PCR of DelLP (FS_13s to FS_15s; 6.4 kb; 11-07-2013)
- Reaction of DelLP
| what | µl |
|---|---|
| Gel extracted fragment LP (11-07-2013) | 2 |
| FS_13_short: (1/10) | 1 |
| FS_15_long: (1/10) | 1 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
| DMSO | 1 |
- Conditions of Del LP
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 2:10 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 2:10 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- The Re-PCR did not work, no bands were visible on the gel.
09-07-2013
Amplification I from FS_12 to FS_13; 2.7 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_12: (1/10) | 1 |
| FS_13: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 40 | |
| 1 | 72 | 7 min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP did not work
- PCR was repeated, Annealing was carried out as touchdown starting from 68°C
Amplification II from FS_12 to FS_13; 2.7 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_12: (1/10) | 1 |
| FS_13short: (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 6 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 6 |
| 8 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 50 | |
| 24 | 98 | 1 |
| 72 | 5 | |
| 72 | 50 | |
| 1 | 72 | 11min |
| 1 | 4 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated with the newly ordered short version of primer FS_13, testing different DMSO settings
10-07-2013
Amplification from FS_12 to FS_13(s); 2.7 kb
- Reaction of DelO-P (2.6 kb)
4 reactions with different conditions: with/without DMSO, short/long Primer FS13
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_12: (1/10) | 1 |
| FS_13(long or short): (1/10) | 1 |
| Phusion Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 6/7 |
- Conditions of Del O-P
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 40 | |
| 1 | 72 | 420 |
| 1 | 12 | inf |
Results:
- None of the Amplification neither with primer FS_13 nor with FS_13short worked out, nevertheless did addition of DMSO change the amplification result
- PCR will be carried out as 2-step in another more precise cycler
- PCR will be set up with as touchdown PCR, annealing starting at 68°C
- Furthermore a modified version of the forward primer will be order. This primer (FS_22) includes a recently in the Del Cluster predicted promotor as well as a likewise predicted ribosome binding site
08-07-2013
Amplification from FS_14 to FS_15; 1.4 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_14: (1/10) | 2.5 |
| FS_15: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 42 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 42 | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
08-07-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(2x50µl)
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_01: (1/10) | 2.5 |
| FS_16: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 14 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 3:20 min | |
| 16 | 98 | 1 |
| 60 | 5 | |
| 72 | 3:20 min | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
19-07-2013
Amplificaction from FS_02 to FS_03; 5.3 kb
- Reaction
2x ~50 µL
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2.5 |
| FS_03: (1/10) | 2.5 |
| Phusion flash Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 19 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelAE did not work since a different cycler was used
20-07-2013
Amplification from FS_02 to FS_03; 5.3 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2.5 |
| FS_03: (1/10) | 2.5 |
| Phusion flash Master Mix | 25 |
| DMSO | 2,5 |
| dd H2O | 19 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelAE worked but a smear occured, therefore bands were cut out carefully and only used for a test restriction digest
15-07-2013
Amplification from FS_04 to FS_07, 11.1 kb
3x 20µl of reaction mix
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 2 |
| FS_07: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 70 ↓ 0.5 | 5 | |
| 72 | 3:00 min | |
| 18 | 98 | 1 |
| 68 | 5 | |
| 72 | 3:00 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
Results:
- Amplification of DelEG worked as expected
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
21-07-2013
Re-PCR of DelEG (FS_04 to FS_07; 11.1 kb; 05-07-2013)
- Reaction
| what | µl |
|---|---|
| Fragment FS_04 to FS_07 amplified 15-07-2013 | 1 |
| FS_04: (1/10) | 2 |
| FS_07: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 70 ↓ 0.5 | 5 | |
| 72 | 3:00 min | |
| 18 | 98 | 1 |
| 68 | 5 | |
| 72 | 3:00 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
Results:
- Re-PCR of DelEG in order to increase specifity did not work
16-07-2013
Amplification from FS_08 to FS_11_short; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_11_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 2:30 min | |
| 24 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
- Conditions II
| Biorad C1000 Touch Block B | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 2:30 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
- Conditions III
| Biometra T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 70 ↓ 0.5 | 5 | |
| 72 | 2:30 min | |
| 18 | 98 | 1 |
| 68 | 5 | |
| 72 | 2:30 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
Results:
- Only a small band with conditions II was visible
- There were no bands with the other two
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
18-07-2013
Amplification from FS_08 to FS_11_short; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_11_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad C1000 Touch | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 64 ↓ 0.5 | 5 | |
| 72 | 2:10 min | |
| 18 | 98 | 1 |
| 60 | 5 | |
| 72 | 2:10 min | |
| 1 | 72 | 10 min |
| 1 | 12 | inf |
Results:
- Amplification of DelG did not work, no product was detectable
- PCR will be repeated at higher temperature to decrease formation of secondary structures and thereby improve binding of primers to the desired sequence
Re-Amplification from FS_08 to FS_11_short; 6.5 kb; 09-07-2013)
- Reaction
| what | µl |
|---|---|
| DelG (09-07-2013) | 4 |
| FS_08 (1/10) | 2 |
| FS_11_short (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 1 |
- Conditions II
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 2:30 | |
| 16 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 | |
| 1 | 72 | 10min |
| 1 | 4 | inf |
Results:
- Amplification of DelG was not sucessful, the Re-PCR did not yield the desired product
- forward Primer will be reused but reverse primer changed, in order to obtain a shorter amplicon and another strategy for the Gibson Assembly
19-07-2013
Amplification from FS_08 to FS_09; 3.3 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_09: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 70 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 6min |
| 1 | 12 | inf |
Results:
- Amplification of DelG did not lead to the desired product, a small band of the desired size was visible and cut for validation and Re-PCR
- Re-PCR will be run
21-07-2013
Re-PCR from FS_08 to FS_09; 3.3 kb; 19-07-2013
- Reaction
| what | µl |
|---|---|
| Fragment FS_08 to FS_09 (19-07-2013) | 1 |
| FS_08: (1/10) | 2 |
| FS_09: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 70 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 6min |
| 1 | 12 | inf |
Results:
- Re-Amplification of DelG did not work
- initial amplification will be repeated at lower annealing temperature
18-07-2013
Amplification from FS_13s to FS_15s; 6.4 kb
- Reaction of DelLP
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_13_short: (1/10) | 2 |
| FS_15_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions of Del LP
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 2:10 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:10 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- The second try to amplify DelL-P did not work. There were no bands visible on the gel.
- It does not make sense to try this again.
17-07-2013
Amplification from FS_22 to FS_13(s); 2.7 kb
4 reactions (with long and short primer FS13 and with conditionI and conditionII)
- Reaction of DelOP
| what | µl |
|---|---|
| D.acidovorans | 1 |
| FS_22: (1/10) | 2 |
| FS_13 (long or short): (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I of DelOP
| Biorad C1000 Touch Block B | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:00 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:00 min | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
- Conditions II of DelOP
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 1:00 min | |
| 1 | 72 | 5:00 min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP failed again
- PCR will be repeated, annealing will be carried out 65°C (touchdown) as primer binding seems not to occure at high temperatues as the ones tested in the last amplification attempts
19-07-2013
Amplification from FS_22 to FS_13s; 2.7 kb
--> reaction mixture with and without DMSO
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 4/5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP failed again, unexpected bands as well as a smear occured
- Annealing temperature will be further decreased, to investigate if amplification of the intended product occurs at lower temperatures
20-07-2013
Amplification from FS_22 to FS_13s; 2.7 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 4 |
- Conditions
| Biorad C1000 Touch Block B | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 60 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP did not work, only unintented products as well as a slight smear occured
- Reaction will be carried out again at lower annealing temperature to allow primer binding to the intended sequences
21-07-2013
Amplification from FS_22 to FS_13s; 2.7 kb
4x 20µl (with, without DMSO; 60touchdown, 72 twostep)
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 4/5 |
- Conditions I
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 60 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 58 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
- Conditions II
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 30 | 98 | 1 |
| 72 | 1:00 min | |
| 1 | 72 | 5:00 min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP led to inconclusive results, a very thin band of the intended length as well as several other bands and a smear occured
- Band were cut out and DNA purified using QIAquick Gel Extraction Kit to verify amplicon in a re-PCR
Re-PCR of DelOP (FS_22 to FS_13s; 2.7 kb; 19-07-2013)
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | - |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated as re-pcr from the previously obtained sample
22-07-2013
Re-PCR of DelOP (FS_22 to FS_13; 2.7 kb; 19-07-2013)
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short 19-07-2013) | 1 |
| FS_22: (1/10) | 2 |
| FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Re-PCR did lead ot a band at the expected size but also a smear and several unexpected bands
- Re-PCR will be repeated
24-07-2013
Re-PCR of DelOP (FS_22 to FS_13; 2.7 kb; 19-07-2013)
3x20µl
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1 |
| FS_22: (1/10) | 2 |
| FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Re-PCR of DelOP did not work, no bands were visible
- Re-PCR will be repeated
26-07-2013
Restriction digest of fragment FS_02 to FS_03; 5.3 kb; 08-07-2013 with EcoRI-HF
Incubation at 37°C for 1 h 45 min
| what | µL |
|---|---|
| FS_02 to FS_03 (08-07-2013) | 15 |
| EcorRI-HF | 0.5 |
| Buffer CutSmart | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 3054, 2260 |
Results:
- restriction digest of DelAE did not work, since incubation time might have been to short
27-07-2013
Amplificaction from FS_02 to FS_03; 5.3 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2.5 |
| FS_03: (1/10) | 2.5 |
| Phusion flash Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 19 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 | |
| 1 | 72 | 12 min |
| 1 | 12 | inf |
Results:
- Amplification of DelAE didnt work out, only smear occured
- repeat PCR with better cycler
Amplificaction from FS_02 to FS_03; 5.3 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1.5/1 |
| FS_02: (1/10) | 2.5/5 |
| FS_03: (1/10) | 2.5/5 |
| Phusion flash Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 19/14 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 2:30 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 2:30 | |
| 1 | 72 | 12 min |
| 1 | 12 | inf |
Results:
- Amplification of DelAE worked with both 200 and 400 nM of Primers, nevertheless amplification was more specific with the higher primer concentration
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
Amplification I from FS_02 to FS_24; 7.1 kb
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 4/2 |
| FS_24: (1/10) | 4/2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 0/4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 3:50 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
- Conditions II
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:50 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:50 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAE worked with 200 nM primer concentration at an annealing temperature of 68°C and 400 nM at an annealing temperature of 65°C, the product obtained at 65°C was more specific
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
Amplification II from FS_02 to FS_24; 7.1 kb
2 reactions, 68°C Touchdown with 200nM Primers and 65°C constant with 400nM Primers
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2/4 |
| FS_24: (1/10) | 2/4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 0/4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 5:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
- Conditions II
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 5:40 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 5:40 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification did not work, neither with 200nM and 68°C touchdown, nor with 400nM and 65°C constant.
- Repeat amplfication with different conditions as primers did not bind very effectively
Amplification III from FS_02 to FS_24; 7.1 kb
2 reactions, 66°C Touchdown with 200nM Primers and 60°C constant with 400nM Primers
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2/4 |
| FS_24: (1/10) | 2/4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 0/4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 60 | 5 | |
| 72 | 5:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
- Conditions II
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 5:40 min | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 5:40 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification from DelAE (7.1 kbp) failed again
- stick to the old strategy and use previously obtained fragments with different other fragments for gibson assembly.
29-07-2013
Restriction digest of FS_02 to FS_03; 5.3 kb;(27-07-2013; II) with BglII
Incubation at 37°C for about 3 h
| what | µL |
|---|---|
| FS_02 to FS_03 (27-07-2013; II) | 15 |
| BglII | 1 |
| Buffer 3.1 | 2 |
| dd H2O | 2 |
Expected fragment lengths: 2,146 kb; 1,862 kb; 1,306 kb
Results:
- Restriction digest shows the expected product sizes
- indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC
26-07-2013
Restriction digest of FS_02 to FS_05; 11.2 kb; 04-07-2013 with EcoRI-HF
Incubation at 37°C for 1hour 45min
| what | µL |
|---|---|
| FS_02 to FS_05 (04-07-2013) | 15 |
| EcoRI-HF | 0.5 |
| Buffer CutSmart | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 4624, 4354, 2260 |
Results:
- restriction digest did not work as expected
- test restriction will be repeat with higher amount of enzyme and longer incubation time
27-07-2013
Amplification of FS_02 to FS_05; 11.2 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2.5 |
| FS_05: (1/10) | 2.5 |
| Phusion flash Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0,5 | 5 | |
| 72 | 3:00 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:00 | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAE worked, but a slight smear as well as several unexpected bands occured
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit but PCR has to be further optimized in order to improve product quality
27-07-2013
Amplification from FS_02 to FS_26; 16.7 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2 |
| FS_26: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 3:50 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work, unspecific band at approximately 3.5 kbp occured
Amplification 1 from FS_02 to FS_23; 22.8 kb
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 4/2 |
| FS_23: (1/10) | 4/2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 0/4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 6:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
- Conditions II
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 6:40 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 6:40 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAG did not work, several unspecific bands occured
Amplification 2 from FS_02 to FS_23; 22.8 kb
4 reactions, 2 with 200nM Primers and 2 with 400nM Primers (both concentrations for each condition)
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 4/2 |
| FS_23: (1/10) | 4/2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 0/4 |
- Conditions I
| Biorad MyCycler, Biometra TProfessional Basic and Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 60 | 5 | |
| 72 | 6:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work, independent of the cycler only unspecific bands at 4.0 and 5.2 kbp occured
27-07-2013
Amplification from DelE (FS_04 to FS_24; 1.6 kb)
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 2 |
| FS_24: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:00 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:00 | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification did not work.
24-07-2013
Restriction digest of fragment FS_04 to FS_07; 11.1 kb with PvuI-HF
Incubation at 37°C for 45 min
| what | µl |
|---|---|
| FS_04 to FS_07 (14-07-2013 and 15-07-2013) | 15 |
| PvuI-HF | 0.8 |
| Buffer CutSmart | 2 |
| dd H2O | 2.8 |
| Expected fragment lengths [bp] | 6187, 4917 |
Results:
- restriction digest did not work
- digest will be repeated with newly amplified and purified DelEG
28-07-2013
Amplification from FS_04 to FS_09 ; 14.4 kb
2 reactions with conditions I and II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_04: (1/10) | 2 |
| FS_09: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 4:40 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
- Conditions II
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 4:40 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 4:40 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
Amplification from FS_26 to FS_07
_FS7to26(68)_FS24to26(65)_FS24to26(68).png)
This amplification did not make sense, two reverse Primer were used. We mixed up Primer FS_24 with Primer FS_26.
2 reactions with conditions I and II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_26: (1/10) | 2 |
| FS_07: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
- Conditions II
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification of DelEG did not work, neither with annealing at a constant temperature of 65°C nor with a touchdown starting from 68°C
- later on it was discovered, that primers had been mixed up
Amplification from FS_26 to FS_24
This amplification did not make sense, two reverse Primer were used.
2 reactions with conditions I and II
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_26: (1/10) | 2 |
| FS_24: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 13 min |
| 1 | 10 | inf |
- Conditions II
| Biorad C1000 Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification of DelEG did not work, neither with annealing at a constant temperature of 65°C nor with a touchdown starting from 68°C
- later on it was discovered, that primers had been mixed up
22-07-2013
Re-PCR from FS_10 to FS_23; 3.3 kb; 13-07-2013
- Reaction
| what | µl |
|---|---|
| Fragment FS_10 to FS_11_short (13-07-2013) | 1 |
| FS_10: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
--> First cycles with wrong program
| Biorad C1000 Touch Block A | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:10 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 1:10 min | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
Results:
- Re-Amplification of DelG lead to the expected fragment but also to a smear, therefore fragment will not be used for Gibson Assembly but restriction digests to validate the product.
Amplification from FS_08 to FS_23; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 4/- |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG worked, leading to the expected product as well one other unexpected band.
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
Amplification from FS_08 to FS_11s; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_11_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 14 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 16 | 98 | 1 |
| 60 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
23-07-2013
Amplification from FS_08 to FS_23; 6.5 kb
_psb6A1(3)_4xFS08toFS23_empty_log2_1.png){kind=spacer}
_psb6A1(3)_4xFS08toFS23_empty_log2_cut_1.png){kind=spacer}
4x 20µl
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_08: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- Gel to proof quality of gel extraction was run
26-07-2013
Restriction digest of Fragment FS_08 to FS_23; 6.5 kb; 23-07-2013 with BglII
Incubation at 37°C for 1hour 45min
| what | µl |
|---|---|
| FS_08 to FS_23 cut1 (23-07-2013) | 15 |
| BglII | 0.5 |
| Buffer 3.1 | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 3291, 1936, 1303 |
Results:
- Restriction digest of DelG (FS_08 to FS_23) was successful though one unexpected band (presumably carry over) appeared
- Construct will be further validated by sequencing before potential Gibson Assembly
25-07-2013
Re-PCR I from FS_22 to FS_13; 2.7 kb; 19-07-2013
3x20µl
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1 |
| FS_22: (1/10) | 4 |
| FS_13_long: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| dd H2O | 1 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP led to a smear and several unexpected bands
- Re-PCR will be repeated
Re-PCR I from FS_22 to FS_13; 2.7 kb; 19-07-2013
_3xFS22toFS13.png)
_3xFS22toFS13_cut.png)
3x20µl
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1 |
| FS_22: (1/10) | 2 |
| FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- a smear as well as a band of the expeceted size occured
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- DNA will be used for restriction digests to validate amplicon
26-07-2013
Restriction digest of fragment FS_22 to FS_13s; 2.7 kb; 21-07-2013 with EcoRI-HF
Incubation at 37°C for 1h 45min
| what | µl |
|---|---|
| FS_22 to FS_13short (21-07-2013) | 20 |
| EcorRI-HF | 0.8 |
| CutSmart | 2.5 |
| dd H2O | 2.5 |
| Expected fragment lengths [bp] | 1883, 960 |
Results:
- Restriction digest did not work, the amplified fragment was either not cut or the fragments are not visible due to a very small amount of DNA
- Re-PCR will be repeated to obtain the higher amounts of DNA required for the restriction digest
30-07-2013
Re-PCR from FS_22 to FS_13; 2.7 kb; 19-07-2013
3x20µl
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1 |
| FS_22: (1/10) | 2 |
| FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated with different primer concentrations to estimate whether primer dimes might be the reason for insufficient amplification
Restriction digest from FS_22 to FS_13; 2.7 kb; 25-07-2013 with EcoRI-HF
Incubation at 37°C for
| what | µl |
|---|---|
| FS_22 to FS_13long(25-07-2013) | 17 |
| EcoRI | 1 |
| Buffer CutSmart | 2 |
Amplification from FS_22 to FS_13s; 2.7 kb
2x20µl
- Reaction
| what | µl |
|---|---|
| D.acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 4 |
| FS_13_short: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| dd H2O | 1 |
20µl
- Reaction
| what | µl |
|---|---|
| D.acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP resulted in a small band atthe desired lenght, but also a smear and several unexpected bands
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- amplicon will be used for restriction digest to validate the construct
Amplification from FS_22 to FS_13(s/l); 2.7 kb
- Reaction
| what | µl |
|---|---|
| D.acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short/FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 4/5 |
- Conditions
| Biorad C1000 Touch | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 70 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
29-07-2013
Restriction digest of fragment FS_02 to FS_03 (5.3 kb; 27-07-2013; II) with BglII
Incubation at 37°C for about 3 h
| what | µL |
|---|---|
| FS_02 to FS_03 (27-07-2013; II) | 15 |
| BglII | 1 |
| Buffer 3.1 | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 2146, 1862, 1306 |
Results:
- Restriction digest shows the expected product sizes
- indicator for correct amplicon but to be sure, PCR product will be prepared for single read sequencing by GATC
29-07-2013
Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 27-07-2013 with BglII
Incubation at 37°C for about 3 hours
| what | µL |
|---|---|
| FS_02 to FS_05 (27-07-2013) | 15 |
| BglII | 1 |
| Buffer 3.1 | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 7229, 2146, 1862 |
Results:
- Test restriction digest worked out as predicted
30-07-2013
Amplification from FS_02 to FS_05; 11.2 kb
2 reactions 200nM/400nM Primers
- Reaction
| what | µL 2nd PCR |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02 (1/10) | 5/2.5 |
| FS_05 (1/10) | 5/2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 11.5/16.5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification did not work properly, as there were several unspecific bands. However we still had product.
30-07-2013
Amplification from FS_02 to FS_26; 16.7 kb
4 reactions, 68°C Touchdown and 65°C constant with 200nM and 400nM Primers
- Reaction
| what | µl |
|---|---|
| D. acidovorans DSM-39 | 1 |
| FS_02: (1/10) | 2/4 |
| FS_26: (1/10) | 2/4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 0/4 |
- Conditions I
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 5:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
- Conditions II
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 5:40 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 5:40 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAG did not work, neither annealing at 65°C constant nor a touchdown starting from 68°C led to the desired product
29-07-2013
Restriction digest of fragment FS_08 to FS_23; 6.5 kb; 23-07-2013 with ClaI
Incubation at 37°C for about 3 hours
| what | µl |
|---|---|
| FS_08 to FS_23 (2) (23-07-2013) | 15 |
| ClaI | 1 |
| Buffer CutSmart | 2 |
| dd H2O | 2 |
| Expected fragment lengths [bp] | 3291, 1936, 1303 |
Results:
- Restriction digest of DelG (FS_08 to FS_23) was successful all predicted bands are clearly visible
30-07-2013
Re-PCR of DelOP FS_22 to FS_13; 2.7 kb; 19-07-2013
3x20µl
- Reaction
| what | µl |
|---|---|
| Fragment FS_22 to FS_13_short (19-07-2013) | 1 |
| FS_22: (1/10) | 2 |
| FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated with different primer concentrations to estimate whether primer dimes might be the reason for insufficient amplification
Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 25-07-2013 with EcoRI-HF
Incubation at 37°C for
| what | µl |
|---|---|
| FS_22 to FS_13long(25-07-2013) | 17 |
| EcoRI | 1 |
| Buffer CutSmart | 2 |
| Expected fragment lengths [bp] | 1883, 960 |
Amplification from FS_22 to FS_13s; 2.7 kb
2x20µl
- Reaction
| what | µl |
|---|---|
| D.acidovorans | 1 |
| FS_22: (1/10) | 4 |
| FS_13_short: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| dd H2O | 1 |
20µl
- Reaction
| what | µl |
|---|---|
| D.acidovorans | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP resulted in a small band atthe desired lenght, but also a smear and several unexpected bands
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- amplicon will be used for restriction digest to validate the construct
Amplification from FS_22 to FS_13(s/l); 2.7 kb
- Reaction
| what | µl |
|---|---|
| D.acidovorans | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short/FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1/- |
| dd H2O | 4/5 |
- Conditions
| Biorad C1000 Touch | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 70 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 12 | inf |
01-08-2013
Amplification from FS_22 to FS_13(s); 2.7 kb
- Reaction
| what | µl |
|---|---|
| D.acidovorans DSM-39 | 1 |
| FS_22: (1/10) | 2 |
| FS_13_short/FS_13_long: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 92 | 5 | |
| 91 | 5 | |
| 90 | 5 | |
| 89 | 5 | |
| 88 | 5 | |
| 87 | 5 | |
| 86 | 5 | |
| 85 | 5 | |
| 84 | 5 | |
| 82 | 5 | |
| 80 | 5 | |
| 78 | 5 | |
| 76 | 5 | |
| 74 | 5 | |
| 72 | 5 | |
| 70 | 5 | |
| 68 | 5 | |
| 66 | 5 | |
| 64 | 5 | |
| 62 | 5 | |
| 60 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Results:
- Amplification of DelOP did not work with stepwise cooling to the intended annealing temperature of 60°C
07-08-2013
Amplification from FS_22 to FS_13; 2.7 kb
_3x14-15(L)besch.png)
_3x14-15(L)cut.png)
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_22: (1/10) | 2 |
| FS_13: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Amplification from FS_22 to FS_13; 2.7 kb
_DelH_1kbbesch.png)
_DelH_1kb_cut.png)
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_22: (1/10) | 2 |
| FS_13: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 55 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP led to a band of the desired size as well as a smear and several different sideproducts
- Band was cut out and DNA purified using QIAquick Gel Extraction Kit to be restriction digested for validation of the PCR product
- Amplification will be repeated at lower temperature to obtain more product
07-08-2013
Sequencing Results
We send the following samples (amplified fragments of D. acidovorans DSM-39 and backbone pSB4K5 from the partsregistry) to GATC for sequencing:
- AE (FS_02-FS_03) with Primer FS_02
- AE (FS_02-FS_03) with Primer FS_03
 Alignment of DelAE(FS_02 to FS_03) with Primer FS_02 |
Alignment of DelAE(FS_02 to FS_03) with Primer FS_03 |
08-08-2013
Amplification from FS_02 to FS_03; 5.3kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 4 |
| FS_03: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 1:30 | |
| 1 | 72 | 7min |
| 1 | 12 | inf |
Results:
- Amplification of DelAE was repeated successfully with the new strain SPH-1
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- if fragment is used in Gibson assembly the amplification has to be repeated to increase the amount of product
07-08-2013
Amplification I from FS_02 to FS_05; 11.2 kb
_FS21-26(FG)_FS8-23(G)besch.png)
_FS21-26(FG)_FS8-23(G)cut.png)
- Reaction
| what | µL |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02 (1/10) | 2.5 |
| FS_05 (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAF did not work
- Repeat amplification in case any errors were made
Amplification II from FS_02 to FS_05; 11.2 kb
_4xpSB4K5_2xFS02-FS05(AF)besch.png)
_4xpSB4K5_2xFS02-FS05(AF)_cut.png)
- Reaction
| what | µL |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02 (1/10) | 2.5 |
| FS_05 (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAF did not work as no product appeared, therefore conditions of PCR have to be optimized
- as extremely long amplicons in the past were usually easier to amplify from agar plate (colony PCR) the same conditions will be tried on the colony as template
08-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
| what | µL |
|---|---|
| D. acidovorans SPH-1 (from agarplate) | 1 |
| FS_02 (1/10) | 2.5 |
| FS_05 (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 4 | inf |
Results:
- Amplification of DelAF did not work as no product appeared, therefore conditions of PCR have to be optimized
- PCR will be repeated with glycerol stock as template
09-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction
| what | µL 2nd PCR |
|---|---|
| D. acidovorans SPH-1 (glycerol stock) | 1 |
| FS_02 (1/10) | 2.5 |
| FS_05 (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 3 min | |
| 18 | 98 | 1 |
| 66 | 5 | |
| 72 | 3 min | |
| 1 | 72 | 10 min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF did not work as no product appeared, therefore conditions of PCR have to be optimized
- PCR will be repeated with a higher annealing temperature as primers might not have bound due to secondary structes
- PCR will be repeated with lower annealing temperatue as primers might not have bound due to high temperatures
10-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
200nM_FS02-05(AF)400nM_1kbruler.png)
200nM_FS02-05(AF)400nM_1kbruler_cut.png)
- Reaction
| Reagent | DelAF | |
|---|---|---|
| Template | D.acidovorans SPH-1 colony | |
| Primer fw | 2 µL FS_02 | 4 µL FS_02 |
| Primer rev | 2 µL FS_05 | 4 µL FS_05 |
| Phusion flash Ready Mix | 10 µL | |
| dd H2O | 1 µL | - |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 3:15 | |
| 1 | 72 | 10 min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF did not work at a temperature of 58°C constant
- as primers might not have been bound at this low temperatue but neither bound at a temperature of 68°C (touchdown or constant), PCR will be repeated at a temperature of 65°C as touchdown and at constant annealing
Amplification II + III from FS_02 to FS_05; 11.2 kb
- Reaction
| Reagent | DelAF II + III | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4.5 µL FS_02 | |||||||||
| Primer rev | 4.5 µL FS_05 | |||||||||
| DMSO | 1 µL | |||||||||
| Phusion Ready Mix | 10 µL | |||||||||
- Conditions
| Biorad MyCycler | ||||||
|---|---|---|---|---|---|---|
| Cycles | temperature [°C] DelAF II | Time | Cycles | temperature [°C] DelAF III | Time | |
| 1 | 98 | 10 s | 1 | 98 | 10 s | |
| 30 | 98 | 1 s | 12 | 98 | 1 s | |
| 65 ↓ 0.5 | 5 s | |||||
| 65 | 5 s | 72 | 3:15 min | |||
| 18 | 98 | 1 s | ||||
| 72 | 3:15 min | 66 | 5 s | |||
| 72 | 3:15 min | |||||
| 1 | 72 | 10 min | 1 | 72 | 10 min | |
| 1 | 10 | inf | 1 | 10 | inf | |
Results:
- Amplification of DelAF from the new strain finally worked, both conditions (65°C constant and 65°C touchdown) led to specific product but the touchdown PCR resulted in a smear
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- PCR will be repeated with a slightly lower temperature with constant annealing to increase yield but avoid the smear which appeared due to the low annealing temperatures used in the last cycles of the touchdown PCR.
11-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
@62%C2%B0Cconst_RePCRDelAG(FS02-FS23)withFS02-11sastemplate_1,10Template_1,20Template_1kbruler.png)
@62%C2%B0Cconst_RePCRDelAG(FS02-FS23)withFS02-11sastemplate_1,10Template_1,20Template_1kbruler_cut.png)
- Reaction
| Reagent | DelAF |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 4.5 µL FS_02 |
| Primer rev | 4.5 µL FS_05 |
| Phusion Ready Mix | 10 µL |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 62 | 5 | |
| 72 | 3:15 | |
| 1 | 72 | 10 min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF worked out though again a smear and several other bands appeared
- bands were cut out very precise to avoid carry over of any unwanted amplicon and DNA purified using QIAquick Gel Extraction Kit
- gradient PCR will be carried out to determine optimal annealing temperature of primers and thereby obtain product of higher specificity suitable for gibson assembly
07-08-2013
Amplification from FS_02 to FS_26; 16.7 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 2 |
| FS_26: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 3:50 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification did not work as expected, nethertheless band was cut out for further amplification with a Re-PCR or test restriction digest.
08-08-2013
Amplification from FS_02 to FS_23; 22.8 kb
- Reaction I
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 4 |
| FS_23: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Reaction II
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_02: (1/10) | 4 |
| FS_13short: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 6:40 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
10-08-2013
Amplification from FS_02 to FS_26; 16.7 kb
Reaction
| Reagent | DelAG | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4.5 µl FS_02 | |||||||||
| Primer rev | 4.5 µl FS_26 | |||||||||
| DMSO | 1 µl | |||||||||
| Phusion Ready Mix | 10 µl | |||||||||
Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 5:20 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 5:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work no product was detectable
- PCR will be repeated with different Primers
Amplification from FS_02 to FS_11_short; 22.8 kb and from FS_02 to FS_23; 22.8 kb
- Reaction
| Reagent | DelAG | |
|---|---|---|
| Template | D.acidovorans SPH-1 colony | |
| Primer fw | 4.5 µl FS_02 | 4.5 µl FS_02 |
| Primer rev | 4.5 µl FS_11_short | 4.5 µl FS_23 |
| DMSO | 1 µl | |
| Phusion Ready Mix | 10 µl | |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 7:20 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 7:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work no product was detectable, therefore a Re-PCR using the amplification of DelAG from 10-08-13 will be conducted
11-08-2013
Re-PCR from FS_02 to FS_11; 22.8 kb; 10-08-2013
- Reaction
| Reagent | DelAG |
|---|---|
| Template | PCR-Product FS02-FS11s from 10-08-2013 |
| Primer fw | 4.5 µl FS_02 |
| Primer rev | 4.5 µl FS_11 |
| Phusion Ready Mix | 10 µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 7:20 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 7:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAG did not work, therefore the template used for the Re-PCR presumably was not the desired amplicon
08-08-2013
Amplification from FS_04 to FS_05; 6 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_04: (1/10) | 4 |
| FS_05: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 58 | 5 | |
| 72 | 1:30 | |
| 1 | 72 | 7min |
| 1 | 12 | inf |
Results:
- Amplification of DelEF worked, though several side products and a smear occured
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
07-08-2013
Amplification from FS_08 to FS_23; 6.5 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_08: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| dd H2O | 4 |
| DMSO | 1 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
Amplification from SR_01 to FS_23; 6.2 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| SR_01: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
08-08-2013
Restriction digest of fragment SR_01 to FS_23; 6.2 kb; 07-08-2013 with BglII
_FS21-FS26(ClaI)_OP(EcoRI).png)
Incubation at room temperature for about 4h and at 37°C for 1 hours
| what | µl |
|---|---|
| SR_01 to FS_23 (07-08-2013) | 20 |
| BglII | 1 |
| Buffer 3.1 | 3 |
| dd H2O | 6 |
| Expected fragment lengths [bp] | 3415, 3115 |
Results:
- Restriction digest of DelG (SR_01 to FS_23) worked out as predicted, PCR-fragment will be sequenced before potential Gibson Assembly
Concentration measurement
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| DelG | SR01-FS23 | 07-08-2013 | 34.9 ng/µl |
| DelG | FS08-FS23 | 07-08-2013 | 25.6 ng/µl |
09-08-2013
Amplification from SR_01 to FS_23; 6.2 kb
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| SR_01: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG did not work
- Amplification will be repeated as the protocol was established and carried out successfully several times before, so a pipetting error might be the reason of the failure
08-08-2013
Amplification from FS_22 to FS_13, 2.7 kb
- Reaktion
| what | µl |
|---|---|
| D. acidovorans SPH-1 (colony-pcr) | 1 |
| FS_22: (1/10) | 4 |
| FS_13long: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 50 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated at a higher annealing temperature of 65°C to increase primer binding
Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 07-08-2013) with EcoRI-HF
Incubation at Room temperature for about 4h and at 37°C for 1 hours
| what | µl |
|---|---|
| SR_22 to FS_13 (07-08-2013) | 20 |
| EcoRI-HF | 1 |
| Buffer CutSmart | 3 |
| dd H2O | 6 |
| Expected fragment lengths [bp] | 1883, 960 |
Results:
- Restriction digest did not work, amplicon was not cut and total amount of DNA was probably too low
- Restriction digest will be repeated as soon as PCR of DelOP works reliable and reproducable with higher amounts of DNA
Concentration measurement OP (07-08-2013)
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| DelOP | FS22-FS13 | 07-08-2013 | 6.1 ng/µl |
09-08-2013
Amplification I from FS_22 to FS_13, 2.7 kb
- Reaktion
| what | µl |
|---|---|
| D. acidovorans SPH-1 (colony-pcr) | 1 |
| FS_22: (1/10) | 4 |
| FS_13long: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 65 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated at a lower annealing temperature of 55°C in case primers were not able to bind at high temperatures
Amplification II from FS_22 to FS_13; 2.7 kb
fromgylcerolstock.png)
wrong Primers were used
- Reaktion
| what | µl |
|---|---|
| D. acidovorans SPH-1 (colony-pcr) | 1 |
| FS_22: (1/10) | 4 |
| FS_13long: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 55 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work as the wrong primers were used
- Amplification will be repeated with different amounts of DMSO
Amplification III from FS_22 to FS_13; 2.7 kb
_OP(-)_OP(%2B).png)
3 samples, one with, two without DMSO (one with much template, one with less template)
- Reaktion
| what | µl |
|---|---|
| D. acidovorans SPH-1 colony | 1 |
| FS_22: (1/10) | 4 |
| FS_13long: (1/10) | 4 |
| Phusion flash Master Mix | 10 |
| DMSO | 0/1 |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 55 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work, neither with lower amount of template, nor with higher, furthermore the DMSO concentration did not influence the PCR for the given annealing temperature
- PCR will be repeated with recently ordered new Primers which bind outside the intended template in the Del-Cluster of D. acidovorans SPH-1 in order to obtain a specific template suited for a nested PCR of DelOP with Primers FS_22 and FS_14l
10-08-2013
Amplification I, nested PCR different Primers; 2.7 kb
- Reaction
| Reagent | DelOP | ||||
|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | ||||
| Primer fw | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_32 |
| Primer rev | 4 µl FS_13l | 4 µl FS_27 | 4 µl FS_28 | 4 µl FS_29 | 4 µl FS_30 |
| Phusion Ready Mix | 10 µl | ||||
| dd H2O | 1 µl | ||||
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 55 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated as touchdown PCR with annealing starting at 68°C and different primer combinations to the template needed for the nested PCR of DelOP with primers FS_22 to FS_13l
Amplification II, nested PCR with different primers; 2.7 kb
_22-13s_22-27_33-13s_33-27_31-29_31-30_32-29_32-30_22-28_22-29.png)
_22-13s_22-27_33-13s_33-27_31-29_31-30_32-29_32-30_22-28_22-29_cut.png)
- Reaction
| Reagent | DelOP | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4 µl FS_22 | 4 µl FS_22 | 4 µl FS_33 | 4 µl FS_33 | 4 µl FS_31 | 4 µl FS_31 | 4 µl FS_32 | 4 µl FS_32 | 4 µl FS_22 | 4 µl FS_22 |
| Primer rev | 4 µl FS_13s | 4 µl FS_27 | 4 µl FS_13s | 4 µl FS_27 | 4 µl FS_29 | 4 µl FS_30 | 4 µl FS_29 | 4 µl FS_30 | 4 µl FS_28 | 4 µl FS_29 |
| DMSO | 1 µl | |||||||||
| Phusion Ready Mix | 10 µl | |||||||||
| dd H2O | 1 µl | |||||||||
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelOP worked with the Primer combinations FS22-FS13short, FS_22-FS_27, FS_33-FS_13short, FS_33-FS_27, FS_32-FS_30 and FS_22-FS28
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- all the obtained PCR products will be used as specific templates for a nested PCR using primers FS_22-FS_13long
Amplification III from FS_22 to FS_13; 2.7 kb
- Reaction
| Reagent | DelOP | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4 µl FS_22 | |||||||||
| Primer rev | 4 µl FS_13l | |||||||||
| DMSO | 1 µl | |||||||||
| Phusion Ready Mix | 10 µl | |||||||||
| dd H2O | 1 µl | |||||||||
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 68 ↓ 0.5 | 5 | |
| 72 | 1:00 | |
| 18 | 98 | 1 |
| 65 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- hope and wait for the nested PCR
11-08-2013
Nested PCR of DelOP-Fragments from 10-08-2013
- Reaction
| Reagent | DelOP | ||||||
|---|---|---|---|---|---|---|---|
| Template | FS22-13s | FS31-30 | FS33-27 | FS22-28 | FS22-13s | FS31-30 | FS33-29 |
| Primer fw | 4.5 µl FS_22 | ||||||
| Primer rev | 4.5 µl FS_13l | ||||||
| DMSO | 1 µl | ||||||
| Phusion Ready Mix | 10 µl | ||||||
- Conditions
| Biometra TProfessional Basic | |||||
|---|---|---|---|---|---|
| Cycles | temperature [°C] Template FS22-13s, FS31-30, FS33-27, FS22-28 | Time | Cycles | temperature [°C] Template FS22-13s, FS31-30, FS33-29, Time | |
| 1 | 98 | 10 s | 1 | 98 | 10 s |
| 30 | 98 | 1 s | 12 | 98 | 1 s |
| 68 ↓ 0.5 | 5 s | ||||
| 58 | 5 s | 72 | 1:00 min | ||
| 18 | 98 | 1 s | |||
| 72 | 1:00 min | 66 | 5 s | ||
| 72 | 1:00 min | ||||
| 1 | 72 | 5 min | 1 | 72 | 5 min |
| 1 | 10 | inf | 1 | 10 | inf |
Results:
- Nested PCR of DelOP did not work, though concentrations of the used template were fine as checked previously
- PCR will be with an annealing temperature of 55°C with the template obtained from the primer combination of FS_22-FS_13short as nested PCR
07-08-2013
Results Sequencing
Send the following samples (amplified fragments of D. acidovorans DSM-39 and backbone pSB4K5 from the partsregistry) to GATC for sequencing:
- DelL (FS_14 to FS_15) with Primer FS_14
- DelL (FS_14 to FS_15) with Primer FS_15
Alignment of DelL(FS_14 to FS_15) with Primer FS_14 |
Alignment of DelL(FS_14 to FS_15) with Primer FS_15 |
Amplification of DelL (FS_14 to FS_15; 1.4 kb)
- Reaction
| what | µl |
|---|---|
| D. acidovorans SPH-1 | 1 |
| FS_14: (1/10) | 2.5 |
| FS_15: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 42 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 42 | |
| 1 | 72 | 12 min |
| 1 | 4 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
08-08-2013
Concentration measurement of DelL
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| DelL | FS14-FS15 | 07-08-2013 | 51 ng/µl |
09-08-2013
Amplification of DelL (FS_14 to FS_15; 1.4 kb)
- Reaction
| what | µl |
|---|---|
| D. acidovorans | 1 |
| FS_14: (1/10) | 2.5 |
| FS_15: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 42 | |
| 18 | 98 | 1 |
| 63 | 5 | |
| 72 | 42 | |
| 1 | 72 | 12 min |
| 1 | 10 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
07-08-2013
Results Sequencing
Send the backbone pSB4K5 from the partsregistry to GATC for sequencing:
- pSB4K5 with Primer FS_01
- pSB4K5 with Primer FS_16
Alignment of pSB4K5(FS_01 to FS_16) with Primer FS_01 |
Alignment of pSB4K5(FS_01 to FS_16) with Primer FS_16 |
Results:
- The sequence is the same as expected and indicated in the partsregistry.
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(2x50µl)
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_01: (1/10) | 2.5 |
| FS_16: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 62 ↓ 0.5 | 5 | |
| 72 | 1:30 min | |
| 18 | 98 | 1 |
| 60 | 5 | |
| 72 | 1:30 min | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
08-08-2013
Concentration measurement
The concentration of the gel purified fragment was measured using a NanoDrop Instrument.
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| pSB4K5 | FS16-FS01 | 07-08-2013 | 55.9 ng/µl |
12-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
gradient_67.5_67.0_66.5_66.0_65.5_3xOP(FS22-13L)gradientfromPCR-product(FS22-13s)72%C2%B0C2step.png)
gradient_67.5_67.0_66.5_66.0_65.5_3xOP(FS22-13L)gradientfromPCR-product(FS22-13s)72%C2%B0C2step_cut.png)
- Reaction
| Reagent | DelAF | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4.5 µL FS_02 | |||||||||
| Primer rev | 4.5 µL FS_05 | |||||||||
| DMSO | 1 µL | |||||||||
| Phusion Ready Mix | 10 µL | |||||||||
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 67.5 - 65.0 (ΔT = 0.5) ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 65.5 - 63.0 (ΔT = 0.5) | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF worked well, gradient displays an optimal annealing temperature of 65.5°C, which will be used for further amplifications
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
13-08-2013
Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 12-08-2013 with EcoRI-HF
Incubation at 37°C for 2 hours
| what | µL |
|---|---|
| FS_02 to FS_05 (12-08-2013) | 20 |
| EcoRI-HF | 1 |
| CutSmart Buffer | 2.5 |
| dd H2O | 1.5 |
Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp
Results:
- Weak bands of about 4.6kbp visible, as well as on of about 2.6kbp
- digest will be repeated using higher concentrations of DNA to clearify results of restriction digest
Amplification from FS_02 to FS_05; 11.2 kb
_AF(Fs02-05)highDMSO_AF(FS02-05)MM_empty_OP2%C2%B5LDMSO_3xOP1%C2%B5LDMSO_2log.png){kind=spacer}
_AF(Fs02-05)highDMSO_AF(FS02-05)MM_empty_OP2%C2%B5LDMSO_3xOP1%C2%B5LDMSO_2log_cut.png){kind=spacer}
- Reaction
1 sample contains 2µL of DMSO
| Reagent | DelAF | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 4.5 µL FS_02 | |||||||||
| Primer rev | 4.5 µL FS_05 | |||||||||
| DMSO | 1 µL | |||||||||
| Phusion Ready Mix | 10 µL | |||||||||
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65.5 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 63.5 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF worked though a slight smear occured, therefore PCR will be repeated on the more precise Biometra TProfessional Basic cylcer again
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
14-08-2013
Concentration measurement (FS_02 to FS_05; 11.2 kb)
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| DelAF | FS02-FS05 | 11-08-2013 | ~10 ng/µL |
| DelAF | FS02-FS05 | 12-08-2013 | 0 ng/µL |
| DelAF | FS02-FS05 | 12-08-2013 | 0 ng/µL |
Results:
- concentrations are not sufficient for gibson assembly
- PCR will be repeated and gel slices of different reactions will be pooled for one gel extraction using QIAquick Gel Extraction Kit to obtain the concentrations needed for gibson assembly
Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 11-08-2013 with EcoRI-HF
Incubation at 37°C for 2 hours 45min
| what | µL |
|---|---|
| FS_02 to FS_05 (11-08-2013) | 18 |
| EcoRI-HF | 1 |
| CutSmart Buffer | 2.5 |
| dd H2O | 3.5 |
Expected fragment sizes: 2.26kbp; 4.62kbp; 4.32kbp
Results:
- One band of about 5kbp and one of 2.5kbp
- no clear result, digest will be repeated with another enzyme (ClaI), as the enzyme used was beyond expiration date
Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 10-08-2013 with ClaI
Incubation at 37°C for 2 hours
| what | µL |
|---|---|
| FS_02 to FS_05 (10-08-2013) | 20 |
| ClaI | 1 |
| CutSmart Buffer | 2.5 |
| dd H2O | 1.5 |
Expected fragment sizes: 6.9kbp, 4.3kbp
Results:
- restriction digest displays the expected fragments, therefore amplification of the desired fragment can be assumed
- PCR product will be prepared for sequencing by GATC to proof amplification of the desired DNA sequence before Gibson Assembly
17-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction of DelAF
6x20 µL
| Reagent | DelAF | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 2.5 µL FS_02 | |||||||||
| Primer rev | 2.5 µL FS_05 | |||||||||
| Phusion Ready Mix | 10 µL | |||||||||
| DMSO | 1 µL | |||||||||
| dd H2O | 4 µL | |||||||||
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65.5 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 63.5 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF worked
- bands were cut out, pooled and DNA purified using QIAquick Gel Extraction Kit to obtain concentrations needed for gibson assembly
18-08-2013
Amplification from FS_02 to FS_05; 11.2 kb
- Reaction of DelAF
6x20 µL
| Reagent | DelAF | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Template | D.acidovorans SPH-1 colony | |||||||||
| Primer fw | 2.5 µL FS_02 | |||||||||
| Primer rev | 2.5 µL FS_05 | |||||||||
| DMSO | 1 µL | |||||||||
| Phusion Ready Mix | 10 µL | |||||||||
| dd H2O | 4 µL | |||||||||
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 65.5 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 63.5 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- Amplification of DelAF worked
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
12-08-2013
Amplification from FS_02 to FS_11; 22.8 kb
_71.8_71.7_71.4_71.0_70.5_70.0_69.5_69.0_68.5_68.1_67.8_67.7besch.png)
- Reaction
| Reagent | DelAG |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 4.5 µl FS_02 |
| Primer rev | 4.5 µl FS_11 |
| DMSO | 1 µl |
| Phusion Ready Mix | 10 µl |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 72.0 - 67.0 (ΔT = 0.5) ↓ 0.5 | 5 | |
| 72 | 7:20 | |
| 18 | 98 | 1 |
| 70.0 - 65.0 (ΔT = 0.5) | 5 | |
| 72 | 7:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
Results:
- none of the amplifications led to yields of DNA sufficient for Gibson Assembly, therefore the used combination of primers seems not to be sufficient for amplification of 22.0 kbp
13-08-2013
Amplification from FS_02 to FS_11_short; 22.8 kb
- Reaction
| Reagent | DelAG |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 4.0 µl FS_02 |
| Primer rev | 4.0 µl FS_11_short |
| DMSO | 2 µl |
| Phusion Ready Mix | 10 µl |
- Conditions
| Biometra TProfessional Basic | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 72.0 - 67.0 (ΔT = 0.5) ↓ 0.5 | 5 | |
| 72 | 7:20 | |
| 18 | 98 | 1 |
| 70.0 - 65.0 (ΔT = 0.5) | 5 | |
| 72 | 7:20 | |
| 1 | 72 | 10min |
| 1 | 10 | inf |
15-08-2013
Amplification from SR_01 to FS_23; 6.2 kb
- Reaction (5x20 µl)
| what | µl |
|---|---|
| D. acidovorans SPH-1 colony | 1 |
| SR_01: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG was successful
- Bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- Amplification will be repeated to increase the concentration for the Gibson Assembly
16-08-2013
Concentration measurement
the fragment was gel purified
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| DelG | SR01-FS23 | 15-08-2013 | 52.6 ng/µl |
| DelG after nucleotide removal | SR01-FS23 | 15-08-2013 | 30 ng/µl |
Amplification from SR_01 to FS_23; 6.2 kb
- Reaction (5x20 µl)
| what | µl |
|---|---|
| D. acidovorans SPH-1 colony | 1 |
| SR_01: (1/10) | 2 |
| FS_23: (1/10) | 2 |
| Phusion flash Master Mix | 10 |
| DMSO | 1 |
| dd H2O | 4 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 12 | 98 | 1 |
| 66 ↓ 0.5 | 5 | |
| 72 | 3:20 | |
| 18 | 98 | 1 |
| 64 | 5 | |
| 72 | 3:20 | |
| 1 | 72 | 10min |
| 1 | 12 | inf |
Results:
- Amplification of DelG was successful
- Bands were cut out and DNA purified using QIAquick Gel Extraction Kit
12-08-2013
Amplification I from FS_22 to FS_13; 2.7 kb
- Reaction
| Reagent | DelOP | |
|---|---|---|
| Template | D.acidovorans SPH-1 colony | 1µl FS_22-FS_13_short 10-08 |
| Primer fw | 2 µl FS_22 | |
| Primer rev | 2 µl FS_13 | |
| Phusion Ready Mix | 10 µl | |
| dd H2O | 5 µl | |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 55 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP did not work
- PCR will be repeated at very high annealing temperatures to ensure that primers with very complex secondary structure such as the Gibson-Primer FS_13long are able to bind
Amplification II and III from FS_22 to FS_13; 2.7 kb
72.png)
72cut.png)
- Reaction
| Reagent | DelOP |
|---|---|
| Template | 1µl FS_22-FS_13_short 10.08 |
| Primer fw | 4 µl FS_22 |
| Primer rev | 4 µl FS_13 |
| Phusion Ready Mix | 10 µl |
| DMSO | 1 µl |
- Conditions
| Biorad MyCycler | ||||||
|---|---|---|---|---|---|---|
| Cycles | temperature [°C] DelOP II | Time | Cycles | temperature [°C] DelOP III | Time | |
| 1 | 98 | 10 s | 1 | 98 | 10 s | |
| 30 | 98 | 1 s | 12 | 98 | 1 s | |
| 74.0 - 70.0 (ΔT = 2.0) ↓ 0.5 | 5 s | |||||
| 72 | 1:00 min | 72 | 3:15 min | |||
| 18 | 98 | 1 s | ||||
| 66 | 5 s | |||||
| 68 / 70 / 72 | 1:00 min | |||||
| 1 | 72 | 10 min | 1 | 72 | 10 min | |
| 1 | 10 | inf | 1 | 10 | inf | |
Results:
- Amplification of DelOP was successful, 2-step PCR with FS_22-FS_13s as template led to expected bands as well as a slight smear and an unintended product
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- PCR will be repeated to obtain more sample
Re-PCR from FS_22 to FS_13; 2.7 kb; 10-08-2013)
3x 20µl
- Reaction
| Reagent | DelOP |
|---|---|
| Template | 1µl FS_22-FS_13_short 10-08-2013 |
| Primer fw | 4 µl FS_22 |
| Primer rev | 4 µl FS_13 |
| Phusion Ready Mix | 10 µl |
| DMSO | 1 µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 1:05 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP was successfull
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
- gradient PCR with annealing at 74°C, 72°C and 70°C will be carried out to increase yield
Amplification V from FS_22 to FS_13; 2.7 kb
76%C2%B0_74.4%C2%B0_73.8%C2%B0_73.1%C2%B0_72.6%C2%B0_MM_72%C2%B0_1kbpbesch.png)
76%C2%B0_74.4%C2%B0_73.8%C2%B0_73.1%C2%B0_72.6%C2%B0_MM_72%C2%B0_1kbpcut.png)
- Reaction
| Reagent | DelOP |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 4.5 µl FS_22 |
| Primer rev | 4.5 µl FS_13 |
| Phusion Ready Mix | 10 µl |
| DMSO | 1 µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 75.0 - 72.0 (ΔT = 0.5) | 1:05 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP was successfull
- The gradient PCR shows that amplification worked best with an annealing temperature of 72.3°C, interestingly this annealing temperature was used for the remaining mastermix of the gradient PCR which included more DMSO than the other samples as mixing of DMSO often is not perfect and therefore the very last sample is of higher DMSO concentration
- Therefore PCR will be repeated with an annealing temperature of 72.3°C and 10% DMSO
13-08-2013
Restriction digest of fragment FS_22 to FS_13; 2.7 kb; 12-08-2013) with EcoRI-HF
Incubation at 37°C for 2 hours
| what | µl |
|---|---|
| FS_22 to FS_13 (12-08-2013) | 20 |
| EcoRI-HF | 1 |
| CutSmart Buffer | 2.5 |
| dd H2O | 1.5 |
| Expected fragment lengths [bp] | 1883, 960 |
Results:
- restriction digest shows the expected bands, therefore validation if PCR amplicon is sufficient for Gibson Assembly
Amplification from FS_22 to FS_13; 2.7 kb
- Reaction
| Reagent | DelOP |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 4 µl FS_22 |
| Primer rev | 4 µl FS_13 |
| Phusion Ready Mix | 10 µl |
| DMSO | 1/2 µl |
| dd H2O | 1/- µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72 | 1:05 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP was successfull with 10% DMSO leading to one specific band of intended size
- PCR will be repeated with 10% DMSO to obtain the amount of product which is necessary for Gibson Assembly and test restriction digest
14-08-2013
Concentration measurement DelOP
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| DelOP | FS22-FS13 | 12-08-2013 | 11 ng/µl |
17-08-2013
Amplification from FS_22 to FS_13; 2.7 kb
6x20 µl
- Reaction
| Reagent | DelOP |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 2.5 µl FS_22 |
| Primer rev | 2.5 µl FS_13 |
| Phusion Ready Mix | 10 µl |
| DMSO | 2 µl |
| dd H2O | 3 µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72.3 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
18-08-2013
Amplification from FS_22 to FS_13; 2.7 kb
6x20 µl
- Reaction
| Reagent | DelOP |
|---|---|
| Template | D.acidovorans SPH-1 colony |
| Primer fw | 2.5 µl FS_22 |
| Primer rev | 2.5 µl FS_13 |
| Phusion Ready Mix | 10 µl |
| DMSO | 2 µl |
| dd H2O | 3 µl |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles | temperature [°C] | Time [s] |
| 1 | 98 | 10 |
| 30 | 98 | 1 |
| 72.3 | 5 | |
| 72 | 1:00 | |
| 1 | 72 | 5 min |
| 1 | 10 | inf |
Results:
- Amplification of DelOP was successful
- bands were cut out and DNA purified using QIAquick Gel Extraction Kit
15-08-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(2x50µl)
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_01: (1/10) | 2.5 |
| FS_16: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 62 | 5 | |
| 72 | 1:30 min | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 15-08-2013) with DpnI
Incubation at 37°C for about 6 hours
| what | µl |
|---|---|
| FS_16 to FS_01 (15-08-2013) | 30 |
| DpnI | 2 |
| CutSmart Buffer | 4 |
| dd H2O | 4 |
Results:
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
16-08-2013
Concentration measurement
The concentration of the gel purified, and DpnI digested fragment was measured using a NanoDrop Instrument.
| Fragment | Primer | Date PCR | Concentration |
|---|---|---|---|
| pSB4K5 DpnI digested | FS01-FS16 | 15-08-2013 | 30 ng/µl |
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(3x50µl)
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_01: (1/10) | 2.5 |
| FS_16: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad T100 | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 62 | 5 | |
| 72 | 1:30 min | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
17-08-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(3x50µl)
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_01: (1/10) | 2.5 |
| FS_16: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 62 | 5 | |
| 72 | 1:30 min | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Results:
- Amplification worked very well, we had bright bands on the right height.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 17-08-2013) with DpnI
Incubation at 37°C for about 6 hours
| what | µl |
|---|---|
| FS_16 to FS_01 (17-08-2013) | 30 |
| DpnI | 2 |
| CutSmart Buffer | 4 |
| dd H2O | 4 |
Results:
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
18-08-2013
Amplification from FS_01 to FS_16; 4.2 kb
- Reaction
(3x50µl)
| what | µl |
|---|---|
| Template pSB4K5 | 1 |
| FS_01: (1/10) | 2.5 |
| FS_16: (1/10) | 2.5 |
| Phusion Master Mix | 25 |
| DMSO | 2.5 |
| dd H2O | 16.5 |
- Conditions
| Biorad MyCycler* | ||
|---|---|---|
| Cycles-PCR | temperature [°C] | Time [s] |
| 1 | 98 | 5 |
| 12 | 98 | 1 |
| 62 | 5 | |
| 72 | 1:30 min | |
| 1 | 72 | 5 min |
| 1 | 8 | inf |
Restriction digest of pSB4K5 (FS_01 to FS_16; 4.2 kb; 18-08-2013) with DpnI
Incubation at 37°C for about 6 hours
| what | µl |
|---|---|
| FS_16 to FS_01 (18-08-2013) | 30 |
| DpnI | 2 |
| CutSmart Buffer | 4 |
| dd H2O | 4 |
Results:
- Digested fragment was run on a gel.
- Bands were cut out and DNA purified using QIAquick Gel Extration Kit.
