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Template:Team:KULeuven/29 July 2009/BlueLightReceptor
From 2009.igem.org
An extra PCR on the MC4100 E.coli colony was performed using the primers 2171 and 2172. This was done to get extra promoter region templates (360bp)
GFP ()
- Miniprepped and nanodropped
- Concentration: 85,6ng/μl
- 260/280: 1,87
- A restriction digest was performed to cut the plasmid with EcoRI and XbaI
- A mixture of 20μl was made(3x):6μl DNA, 2μl bufferH, 1μl EcoRI and 1μl XbaI, 10μl AD
- The mixture was incubated for at least an hour at 37°C
BLR promoter region
The PCR product that was purified friday (24/07) is digested with EcoRI and partially digested with SpeI
- Digestion with EcoRI
- Following mixture was made (x6): 5μl DNA, 2μl buffer H, 1μl EcoRI, 12μl MilliQ
- Incubated for 1h at 37°C
- Partial digestion with SpeI
- Dilution of the enzymes:
- AD/b: 225μl MQ + 25μl buffer H
- 1/100: 1μl SpeI + 99μl AD/b
- 1/200: 50μl 1/100 + 50μl AD/b
- 1/500: 20μl 1/100 + 80μl AD/b
- Made following mixture:
- Dilution of the enzymes:
| I | II | III | IV | V | VI | |
|---|---|---|---|---|---|---|
| EcoRI digestion mix | 20μl | 20μl | 20μl | 20μl | 20μl | 20μl |
| diluted SpeI | 1μl 1/200 | 2μl 1/200 | 3μl 1/200 | 1μl 1/500 | 2μl 1/500 | 3μl 1/500 |
| AD | 4μl | 3μl | 2μl | 4μl | 3μl | 2μl |
- The mixture was incubated for 15 min at 37°C
After the restriction digests, the products had to be checked for their length. So, an agarose gel was run for both the cut GFP-plasmids and the cut promoter region. A photograph was taken and following conclusions were made:
- Plasmids with GFP appeared to have cut decently
- Promoter region: the partial digestion did not seem to have done the trick. We only found a lane around 360 bp while we expected to find another lane just under 200 bp due to cutting at the "forbidden" SpeI site in the middle. As we did not find this lane in our gel we concluded that SpeI probably did not cut. This might be because it was diluted too much or because we did not incubate long enough.
We concluded to purify both the plasmids and the promoter region through gel extraction. After nanodropping, we had these results:
- Plasmids:
- Concentration: 22ng/µl
- 260/280: 1,83
- Promoter region (two samples):
- Concentration: 31,7ng/µl and 32,7ng/µl
- 260/280: 1,84 and 2,06
The conclusion of the day was to redo a partial digestion on the promoter regions, this time under different conditions (longer incubation time and less diluted). At the same time a Knelow technique would be used on the newly PCR-ed promoter region in order to cut out the SpeI site.
Later that evening we received an email from Regine Hengge (co-author on the article [http://www.ncbi.nlm.nih.gov/pubmed/19240136 The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli]). This contained valuable information about the location of the actual promoter in our purified region.
