Team:UCLA/Notebook/Spider Silk Genetics/1 September 2015
9/1/2015
Miniprep
- Used qiagen kit to prep M1-12(1C3)/(T7) and M1/2[1:2]-12(1C3)/(T7)
- Sent 3 samples of each for sequencing (Fasih prepped)
ICA for M1-15
- Used established protocol.
- Put the M1-SeqAB2 in the fourth position (the second AB type monomer).
PCR for M1-15
- Set up 2x 50 uL reactions.
| Volume (uL) | |
|---|---|
| 5x Q5 Buffer | 10 |
| 10 mM dNTPs | 1 |
| 10 uM For (F-03) | 2.5 |
| 10 uM Rev (G-03) | 2.5 |
| Template | 1 |
| Q5 Polymerase | 0.5 |
| ddH2O | 32.5 |
| Total | 50 uL |
| 98 C | 30 sec |
| 98 C | 10 sec |
| 66 C | 20 sec |
| 72 C | 40 sec |
| repeat from step 2 | 20x |
| 72 C | 2 min |
| 12 C | hold |
- Visualized on 1% TAE gel with 2 uL of 1 kb ladder.
- The indicated band was excised for extraction.
- From a visual inspection, the yield is somewhat lower than that obtained for the 12-mers. This is probably due to reduced efficiency when using ICA to construct longer chains.