Team:Cambridge/LabBook/Week10
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Week 10: Monday 13th September - Sunday 19th September
Monday
100. Expt: Adding pBAD to P.P. pSB1C3 (Hannah and Emily)
- Took P.P. pSB1C3 out of fridge
- Extracted pBAD
- Restricted pBAD with E + S (using protocol from Fermentas below)
- Restricted P.P. pSB1C3
- Ran on gel
Here are gel columns:
Lane 1 | Lane 2 | Lane 3 | Lane 4 | Lane 5 | Lane 6 | Lane 7 |
EZ Ladder II | P.P.pSB1C3 | P.P.pSB1C3 | pBAD (1) | pBAD (1) | pBAD (2) | pBAD (2) |
5µl | 24µl | 12µl | 12µl | 12µl | 12µl | 12µl |
Results of gel: P.P.+pSB1C3 worked but pBAD could not be seen. P.P.+pSB1C3 was around 5kb as expected. Ben performed gel extraction on this.
We used pBAD and P.P.+pSB1C3 from a previous experiment (from the freezer) and performed ligation using the Fermentas protocol below, but using the following quantities:
Volume (µl) | |
P.P. pSB1C3 | 1 |
pBAD | 1 |
DI H20 | 13 |
5x Rapid ligation Buffer | 4 |
T4 DNA Ligase | 1 |
20 |
Fermentas Restriction Digest Protocol
1. Combine the following reaction components at room temperature in the order indicated:
Plasmid DNA | |
Water, nuclease-free | 15µl |
10x FastDigest Buffer | 2µl |
DNA | 2µl (up to 1µg) |
FastDigest enzyme | 1µl |
20µl |
µl°C