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Team:SDU-Denmark/labnotes9
From 2010.igem.org
Lab notes (9/6 - 9/12)
Contents |
Flagella Group
Restriction Digest of Gel Extracted FlhDCmut, pSB1C3 and pSB1AK3
Date: 9/12 2010
Done By: Sheila
Protocol: RD1.1
Notes: The restriction digests are made in preperation for two different ligations, the first is the ligation of FlhDCmut into the submission backbone; pSB1C3. The second is the the ligation of FlhDCmut into pSB1AK3. Different restriction enzymes were used in, as shown in the following tables
Restriction Digest mixtures
FlhDCmut
| H2O | 48μL |
| EcoR1 | 4μL |
| Pst1 | 4μL |
| FD Green buffer | 8μL |
| Sample | 20μL |
pSB1C3
| H2O | 24μL |
| EcoR1 | 2μL |
| Pst1 | 2μL |
| FD Green buffer | 4μL |
| Sample | 10μL |
FlhDCmut and pSB1AK3 Restriction digest
FlhDCmut
| H2O | 48μL |
| EcoR1 | 4μL |
| Spe1 | 4μL |
| FD Green buffer | 8μL |
| Sample | 20μL |
pSB1AK3
| H2O | 24μL |
| EcoR1 | 2μL |
| Xba1 | 2μL |
| FD Green buffer | 4μL |
| Sample | 10μL |
Following digestion, the samples were loaded and run on a 1.5% agarose gel, before being extracted prior to ligation.
Results:
Ligation of FlhDCmut and pSB1C3
Date: 9/12 2010
Done By: Sheila
Protocol: LG1.2
Notes:
Results:
Ligation of FlhDCmut and pSB1AK3
Date: 9/12 2010
Done By: Sheila
Protocol: LG1.2
Notes:
Results:
Photosensor group
Taq gradient PCR of pKJ606 with PS primers
Date: 9/7 2010
Done By: Maria and Lc
Protocol: CP1.3
Notes:
A gradient PCR was carried out to determine the appropiate annealing temperature of the new PS primers. PCR tubes were marked PSII A-H.
Premix x 9
| Taq buffer (10x) | 22.5uL |
| MgCl2 | 9uL |
| PSII fw primer | 9uL |
| PSII rv primer | 9uL |
| dNTP mix | 4.5uL |
| H20 | 158.5uL |
| Taq polymerase | 1uL |
miniprep of pKJ606 was used as template. 1uL was distrubuted into each tube.
PCR program:
| start | 95C | 2min |
| denaturating | 95C | 1min |
| annealing | se additional table | 1min |
| elongation | 72C | 2min |
| go to | 2 | rep.29x |
| end | 72C | 5min |
| hold | 4C |
Anealing temperatures:
| 56.5C |
| 58.3C |
| 60.7C |
| 63.3C |
| 66C |
| 68.6C |
| 71C |
| 73C |
PCR product was loaded onto a 1.5 agarose gel. gene ruler DNA ladder mix (red) was used as marker.
Results:
Analysis:
Only very weak bands was observed at app. 2000 bp, and very strong bands were observed at app. 4000 bp, indicating that too much template was used in the PCR. An additional PCR was carried out using diluted miniprep as template.
--Tipi 10:27, 21 September 2010 (UTC)
Insertion of PS in pSB1C3 and pSB1AK3
Date: 9/8 - 9/12 2010
Done By: Maria and Lc
Protocol: CP1.1DE1.3RD1.1LG1.2CC1.1TR1.1CP1.3
Pfu PCR amplification of PS (no.1)
Date: 9/8 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
5 PCR reactions are prepared. 2uL Miniprep of pKJ606 are diluted in 8uL H2O to reach a 5x dilution, and are used as template. PCR tubes are marked PSIII A-E.
Premix x6:
| pfu buffer + MgSO4 | 30uL |
| dNTP mix | 9uL |
| PSII fw primer | 9uL |
| PSII rv primer | 9uL |
| H20 | 230uL |
| pfu polymerase | 2.5uL |
| pKJ606 miniprep (5x diluted) | 10uL |
PCR program:
| start | 94C | 3min |
| denaturating | 94C | 2min |
| annealing | 68C | 30s |
| elongation | 72C | 2min30s |
| go to | 2 | rep.29x |
| end | 72C | 5min |
| hold | 4C |
All of the PCR product was loaded onto a 1.5 agarose extraction gel. Gene ruler DNA ladder mix was used ad marker.
Results:
Analysis:
No bands were visible in the gel, indicating that the PCR reaction had been unsuccessfull. This could be due to the low elongation time, wherefore another PCR reaction with a longer elongation time was carried out.
Pfu PCR amplification of PS (no.2)
Date: 9/9 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
5 PCR reactions are prepared. 2uL Miniprep of pKJ606 are diluted in 8uL H2O to reach a 5x dilution, and are used as template. PCR tubes are marked PSIV A-E.
Premix x6:
| pfu buffer + MgSO4 | 30uL |
| dNTP mix | 9uL |
| PSII fw primer | 9uL |
| PSII rv primer | 9uL |
| H20 | 228uL |
| pfu polymerase | 2.5uL |
| pKJ606 miniprep (5x diluted) | 12uL |
PCR program:
| start | 94C | 3min |
| denaturating | 94C | 2min |
| annealing | 68C | 30s |
| elongation | 72C | 4min20s |
| go to | 2 | rep.29x |
| end | 72C | 5min |
| hold | 4C |
All of the PCR product was loaded onto a 1.5 agarose extraction gel. Gene ruler DNA ladder mix was used ad marker.
Results:
Analysis:
Bands were observed at app. 2000bp and bands were extracted by gel extraction
Gel extraction of PS
Date: 9/9 2010
Done By: Maria and Lc
Protocol:DE1.3
Notes:
DNA was extracted from gel according to protocol and each sample was diluted in 20uL.
Analysis:
each sample had a concentration of app. 7ng/uL. All 4 samples were pooled and used for restriction digest.
Restriction digest of PS, pSB1C3 and pSB1AK3
Date: 9/9 2010
Done By: Maria and Lc
Protocol:RD1.1DE1.3
Notes:
Restriction mixture pSB1C3:
| H2O | 24uL |
| FD green buffer | 4uL |
| EcoRI | 2uL |
| PstI | 2uL |
| pSB1C3 | 10uL |
Restriction mixture pSB1AK3:
| H2O | 24uL |
| FD green buffer | 4uL |
| EcoRI | 2uL |
| XbaI | 2uL |
| pSB1AK3 | 10uL |
Restriction mixture PS (used in pSB1C3):
| H2O | 38uL |
| FD green buffer | 8uL |
| EcoRI | 4uL |
| PstI | 4uL |
| PS | 30uL |
Restriction mixture PS (used in pSB1AK3):
| H2O | 38uL |
| FD green buffer | 8uL |
| EcoRI | 4uL |
| SpeI | 4uL |
| PS | 30uL |
Digested samples were loaded onto a 1.5% agarose extraction gel. Uncut PS, pSB1C3 and pSB1AK3 were used as controles. Gene ruler DNA ladder mix was used as marker.
Loading scheme:
| Lane | sample |
| 1 | marker |
| 2 | PS (used in pSB1C3) |
| 3 | uncut PS |
| 4 | PS (used in pSB1AK3) |
| 5 | uncut pSB1C3 |
| 6 | pSB1C3 |
| 7 | uncut pSB1AK3 |
| 8 | pSB1AK3 |
DNA was extracted from gel according to protocol.
Results:
DNA conc:
| sample | conc. (ng/uL) |
| PS (used in pSB1C3) | 5.5 |
| PS (used in pSB1AK3) | 4.2 |
| pSB1C3 | 6.3 |
| pSB1AK3 | 14.3 |
Analysis:
the purified DNA was used for ligation.
