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Team:Heidelberg/Indigoidine
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Revision as of 00:32, 5 October 2013
Project
Notebook
Human Practice
Indigoidine. Proving Modularity of NRPS by Shuffling Domains.
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 5
At the beginning of our wetlab phase, we wanted to transform E. coli cells with plasmids containing an indigoidine synthetase and a 4'-Phosphopanthetheinyl-transferase (PPTase) to see whether we can observe blue colonies on our plates as this has been reported by groups working with indigoidine synthetases (<bib id="Takahashi2007"/><bib id="Brachmann2012"/>).
We focused on the work of Marius Müller et. al. in 2012 (<bib id="Muller2012"/>). The group used the bpsA indigoidine synthetase (blue pigment synthetase A) from S. lavendulae ATCC11924 (<bib id="Takahashi2007"/>) and the PPTase svp from S. verticillus ATCC15003 (<bib id="Sanchez2001"/>) to establish both a fluorescence and a chromophore based reporter assay for mammalian cells by expression of bpsA and svp which results in production of a blue pigment/ fluorophore. The group kindly supported us by sending two of their constructs, namely the pET derived expression vectors pMM64 carrying the bpsA indigoidine synthetase with an ampicillin resistance gene and pMM65 carrying svp and a kanamycin resistance gene. Both bpsA and svp rom the Fussenegger lab are codon-optimized versions for expression in eukaryotic cell lines.
We transformed competent E. coli TOP10 with each plasmid to prepare plasmid DNA and perform a cotransformation of an E. coli Rosetta strain with both plasmids. Transformed cells which carry both plasmids should produce the blue pigment indigoidine.
Unfortunately, there were no blue colonies after the co-transformation. We repeated the experiment under various growth conditions; i.e. we used different incubation temperatures, light conditions, shaking and addition of ascorbic acid, which was reported to stabilize indigoidine in liquid cultures (<bib id="Muller2012"/>).
Week 6
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 7
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 8
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 9
In order to transfer the gold precipitating NRPS from D. acidovorans to E.coli, the necessary modules will be amplified from the D. acidovorans genome and assembled as plasmids. Due to its large size of 18 Kb, the module DelH will be expressed on a separate plasmid. A strategy was developed, primers designed accordingly and necessary BioBricks obtained from the distribution. The D. acidovorans was obtained from the DSMZ and cultured in Acidovorax complex medium.
Week 10
In order to clone the Delftibactin cluster from D. Acidovorans we decided to use Gibson cloning. Accordingly Gibson Primers were designed to amplify our target backbone pSB4K5 with an overlap to DelA. Furthermore the Gibson Primer designed to join DelOP with DelAF will introduce the ribosome binding site BBa_BNILS. Accordingly the Gibson Primer designed to join DelL with DelOP will introduce the ribosome binding site BBa_BAGAINNILSYOUNOOB.
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
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 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
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
