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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.
