Team:UCLA/Notebook/Spider Silk Genetics/4 August 2015
Contents
8/4/2015
PCR: Amplify ICA M1-12 from 08/03/2015
| 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 |
| 98 C | 30 sec |
| 98 C | 10 sec |
| 66 C | 20 sec |
| 72 C | 35 sec |
| repeat from step 2 | 20x |
| 72 C | 2 min |
| 12 C | hold |
Ran PCR product on 1% gel with 1kb ladder
Expected size of a 12-mer: ~1300bp
Gel purified the correct band but yield was too low (~5ng/uL in 15uL) for cloning
Need to redo PCR
PCR: Add End Extensions to M1 Seq Core 1 and 2
| M1 Seq Core 1 | M1 Seq Core 2 | |
| 5x Q5 Buffer | 10 uL | 10 uL |
| 10 mM dNTPs | 1 uL | 1 uL |
| 10 uM Primer 1A-F | 2.5 uL | 2.5 uL |
| 10 uM Primer 1B-R | 2.5 uL | 2.5 uL |
| Template | 0.4 uL | 1.8 uL |
| Q5 Polymerase | 0.5 uL | 0.5 uL |
| ddH2O | 23.1 uL | 21.7 uL |
| Total | 50 uL | 50 uL |
| 98 C | 30 sec |
| 98 C | 10 sec |
| 64 C | 15 sec |
| 72 C | 15 sec |
| repeat from step 2 | 25x |
| 72 C | 2 min |
| 12 C | hold |
Ran samples on 1% gel with 50bp ladder.
Expected size of M1SeqAB is 170bp
Both 1 and 2 showed band of the right size
Samples were PCR purified
Colony PCR for M2-15(T7) and M19(psb1C3)
| 2X Taq Red Master Mix | 6.25 uL |
| 10uM Vf Primer | 0.625 uL |
| Template | 1uL |
| ddH2O | 4 uL |
Ran samples on 1% gel with NEB 1kb ladder
Bands of expected sizes were dim but still present. Picked three of each for sequencing.