"
Team:Heidelberg/Delftibactin/Delftibactin
From 2013.igem.org
Project
Notebook
Human Practice
Delftibactin. Bringing it all together.
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Week 1
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 2
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 3
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 4
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 5
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 6
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 7
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 8
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 9
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 10
This week the project DelRest was launched. In order to clone the Delftibactin cluster from D. Acidovorans into E.coli we decided to use Gibson cloning. Therefore Gibson primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson primer connecting DelOP and DelAG introduces the ribosome binding site BBa_BNILS. Accordingly the Gibson primer for joining DelL with DelOP includes the ribosome binding site BBa_BNILS. The very last gibson primer used to amplify DelL consequently has an overlap to the beginning of the mRFP reporter gene connected to pSB4K5. Check out our vectormap if you are curious about the detailed primer design. The goal of this week was to amplify our previously assembled backbone with the intended overlaps as well as the desired genes from our host organism D. Acidovorans .
Week 11
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.
At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
Week 12
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.
At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed. After the Transformation to DH10β cells and screening by restriction digest we could send samples for the Dipeptide and Tetrapeptide I to sequencing and obtained a positive alignment. Hence we transformed BAP I cells with the positive constructs. The same was...
Week 13
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 14
At the beginning of the week, we could verify that the Gibson Assembly for Tripeptide I was indeed positive, however, the other Gibson Assemblies did not work properly. Instead of picking new colonies, we decided to optimize the Gibson recipe instead, as backbone religations were the most common problem. With these improved protocols, we used Gibson Assembly for the Dipeptide, Tripeptide II and Tetrapeptide I, later that week, Tetrapeptide II followed.
Week 15
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 16
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 17
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 18
dumm dummy
Week 19
After successful dumm dummy
Week 20
dumm dummy
Week 21
dummy
Week 22
dummmmmy
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.
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
