"
Team:SDU-Denmark/labnotes4
From 2010.igem.org
Lab notes (8/2 - 8/8)
Contents |
Group: Photosensor
Restriction digest of PUC19 and the photosensor in pKJ606 with Nde1
Start date: 2/08 End date: 2/08
Methods: Restriction digest, gel electrophoresis
Protocol:RD1.1
Experiment done by: LC
Notes: Since all PCR attempts with VF2 and VR had failed until now, we wanted to see if there was an insert at all in the physical DNA we received. Therefore we cut PUC19 and the photosensor to compare their lengths to each other, since the photosensor should be longer than PUC19 uncut and the cut photosensor should consist of two pieces, a 900 - 1000 BP piece and a 3600 BP piece.
Loading order:
1: PUC19 uncut
2: PUC19 cut
3: Photosensor uncut
4: Photosensor cut
Results:
Analysis:
We can clearly see that the photosensor uncut is longer than the uncut PUC19. We can also see multiple bands on the cut photosensor, which are the expected lengths. This indicates that the photosensor gene should be inserted between VF2 and VR, so we are still unsure why the PCR is not working.
Dreamtaq Green PCR Master Mix (2X) PCR on photosensor with VF2 and VR
Start date: 2/08 End date: 2/08
Methods: PCR, gel electrophoresis
Protocol: Dream TAQ Green PCR Master Mix protocol (modified)
Experiment done by: LC
Notes: Instead of a 50 µl reaction we ran a 25 µl reaction. It was mixed like this:
| Dream TAQ Master
Mix |
12,5 µl |
| VF2 |
1 µl |
| VR |
1 µl |
| Template |
0,5 µl |
| Water |
10 µl |
We used a miniprep of the photosensor as template DNA, hence the small amount of template. The PCR program used was as follows:
| Step |
Temperature |
Time |
Number of cycles |
| Initial denaturation |
95° |
1 - 3 min |
1 |
| Denaturation |
95° |
1 min |
30 |
| Annealing |
55° |
1 min |
30 |
| Extension |
72° |
3 min |
30 |
| Final extension |
72° |
3 min |
1 |
Results:
Analysis:
We got bands at 270 BP, 850 BP (the distance between VF2 and VR without insert) and two bands very high up in the gel (probably the template which did not get amplified and could not run through the 1,5% gel).
