Team:UCLA/Notebook/Spider Silk Genetics/4 September 2015
Contents
9/4/2015
Miniprep for M1-15(1C3)/(T7) and Tamura(1C3)
- Used qiagen kit.
- Elute in 30 uL.
Restriction Digest Test
- Digest with X,P to see insert size.
- Digest 1 ug of plasmid in a 50 uL reaction with 1 uL of each enzyme.
- Digest at 37 C for 1 hr 15 min, heat kill at 80 C for 15 min.
- Visualize on 1% TAE gel with 2 uL of 1 kb ladder.
- M1-15 colonies are wrong; don't send for sequencing.
- Send Tamura(1C3) for sequencing with VF, VR primers.
- Also send old Tamura(1A3) for sequencing as well.
Colony PCR for M1-15(T7)
- Since no colonies are left on M1-15(1C3), will focus on M1-15(T7).
- Picked 8 colonies from M1-15(T7) plate, resuspended in 50 uL LB.
- Colony PCR with 1 uL of resuspended colony as template.
Volume (uL) | |
---|---|
2x Taq Red Master Mix | 6.25 |
10 uM For (VF) | 0.625 |
10 uM Rev (VR) | 0.625 |
Colony | 1 |
ddH2O | 4 |
Total | 12.5 |
95 C | 3 min |
95 C | 25 sec |
56 C | 30 sec |
72 C | 1.5 min |
repeat from step 2 | 30x |
72 C | 5 min |
12 C | hold |
- Visualize colony PCR results on 1% TAE gel with 2 uL of 1 kb ladder.
- Colony PCR shows that the 8 colonies picked are incorrect. Will pick more.