Team:UCLA/Notebook/Spider Silk Genetics/27 July 2015
Contents
7/27/2015
Transformation Results
- 5 colonies on the M2-12(1C3) plate
Colony PCR
- For M2-12(1C3) colonies
- Resuspend colony in 50 uL LB.
- Use 1 uL as a template in 2x Taq Red PCR.
Volume (uL) | |
---|---|
2x Taq Red | 12.5 |
10 uM For (VF) | 1.25 |
10 uM Rev (VR) | 1.25 |
Template (1:50) | 1 uL |
ddH2O | 9 uL |
Total | 25 uL |
95 C | 3 min |
95 C | 25 sec |
56 C | 30 sec |
72 C | 45 sec |
repeat from step 2 | 30x |
72 C | 5 min |
12 C | hold |
PCR Amplification of 15-mer
- Set up 2x 50 uL Reactions for amplification using M2-15mer template.
- Scaled up reactions from 7/24/2015.
- Same conditions, but with 23 cycles instead of 20 cycles.
- Proceed to gel purification.
Results
- Colony number 1 popped open in incubator, and could not be salvaged
- Cast 1% TAE gel. Ran with 2 uL of NEB 1kb ladder.
- Colony PCR indicates that colonies 2, 3 may be the correct size. However, the bands are slightly off shifted from each other. This indicates that one of the samples is probably wrong.
- Will grow 5 mL cultures for colonies 1,2,3 to send for sequencing.
- The M2-15mer PCR is inefficient compared to previous results. We may need to optimize, or just brute force it.
- Excised the band corresponding to the M2-15 for gel purification.
Bacteria Culture
- Set up 1x 100 mL culture each for M1-AB, BC, CA
- Set up 5 mL cultures for colonies M2-12(1C3) colonies 1, 2, 3.