Team:UCLA/Notebook/Spider Silk Genetics/14 August 2015
Contents
08/14/2015
Miniprep samples from yesterday
Sequencing results:
M1-9(T7)
- Colony 1,2 and 4 have the correct sequence
PCR amplify M1-12
Since previous PCRs using ICA elution as template did not work very well, first M1-12 PCR product was also tested as a template
| ICA elution | PCR product | |
| Q5 buff | 10 uL | 10 uL |
| dNTPs | 1 uL | 1 uL |
| F-03 | 2.5 uL | 2.5 uL |
| G-03 | 2.5 uL | 2.5 uL |
| ddH2O | 32.5 uL | 33.3 uL |
| Template | 1 uL | 0.2 uL |
| Q5 Polymerase | 0.5 uL | 0.5 uL |
| 98C | 30 sec |
| 98C | 10 sec |
| 66C | 20 sec |
| x23 | |
| 72C | 45 sec |
| 72C | 2 min |
| 12C | Hold |
Ran samples on 1% gel
Sample 1 with had no band Sample 2 had a very bright band close to the expected size
Maybe it was the ICA elution that was no longer in good condition?
Gel purified the band from sample 2
- 39.69 ng/uL; 260/280: 2.47
Double Digestion of M1-12 PCR product with EcoRI and PstI
| EcoRI | 1 uL |
| Pst I | 1 uL |
| Template | 11 uL |
| Buffer 2.1 | 5 uL |
| ddH2) | 32 uL |
37C for 1.5 hr, 65C for 20 min