Template:Team:KULeuven/29 July 2009/BlueLightReceptor

From 2009.igem.org

(Difference between revisions)

Latest revision as of 09:50, 4 September 2009

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:

	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.

@@ Line 1: / Line 1: @@
-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)
+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 ({{kulpart|BBa_E0240}})'''
-*miniprepped and nanodropped
+*Miniprepped and nanodropped
-**concentration: 85,6ng/μl
+**Concentration: 85,6ng/μl
 **260/280: 1,87
-*a restriction digest was performed to cut the plasmid with EcoRI and XbaI
+*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
+**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
+**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
+*Digestion with EcoRI
-**following mixture was made (x6): 5μl DNA, 2μl bufferH, 1μl EcoRI, 12μl MilliQ
+**Following mixture was made (x6): 5μl DNA, 2μl buffer H, 1μl EcoRI, 12μl MilliQ
-**incubated for 1h at 37°C
+**Incubated for 1h at 37°C
-*partial digestion with SpeI
+*Partial digestion with SpeI
-**dilution of the enzymes:
+**Dilution of the enzymes:
-***AD/b: 225μl MQ + 25μl bufferH
+***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
-**making following mixture:
+**Made following mixture:
 {| border="0" class="Generic"
@@ Line 43: / Line 43: @@
 |-
 |}
-:*the mixture was incubated for 15 min at 37°C
+:*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:
+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
+*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 we diluted it to much or because we did not incubate long enough.
+*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.
+We concluded to purify both the plasmids and the promoter region through gel extraction.
-after nanodropping them we had these results:
+After nanodropping, we had these results:
-*plasmids:
+*Plasmids:
-**concentration: 22ng/µl
+**Concentration: 22ng/µl
 **260/280: 1,83
-*promoter region (two samples):
+*Promoter region (two samples):
-**concentration: 31,7ng/µl and 32,7ng/µl
+**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 incubating time and less diluted). at the same time a Knelow technique would be used on the newly PCRed promoter region in order to cut out the SpeI site.
+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 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]) was recived. This email contained valuable information about the location of the actual promoter in our purified region.
+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.