Team:UCLA/Notebook/Spider Silk Genetics/28 April 2015
4/28/2015
1 Post-Elution Primer PCR Amplification of MaSp2 Sequences
We used the post-elution primers to test amplify 2AB plasmid and to determine an appropriate annealing temperature. We used the following protocol:
| 1x Reaction (uL) | 4x Reaction (uL) | |
| 5x Q5 Buffer | 5 | 20 |
| 10 mM dNTPs | 0.5 | 2 |
| 10 uM For (F-03) | 1.25 | 5 |
| 10 uM Rev (G-03) | 1.25 | 5 |
| 2AB (500 pg) | 1.74 | 6.96 |
| 5x GC buffer | 5 | 20 |
| Q5 Pol | 0.25 | 1 uL |
| ddH2O | 10.01 | 40.04 |
| Total | 25 | 100 |
| 98 C | 30 sec |
| 98 C | 10 sec |
| 69, 66.8, 65.4, 63 C | 15 sec |
| 72 C | 15 sec |
| Repeat from step 2 | 30 x |
| 72 C | 2 min |
| 12 C | hold |
2 Results
Figure 1: Annealing temperature test with MaSp 2AB at different
temperatures. Our expected product is 170 bp.
∙ Base on these results, we decided to scale up to 3x 50 uL reactions for each monomer, and would use 66 C as the annealing temperature.
3 Amplification Again
We scaled up the above protocol for 3x 50 uL reactions for each monomer.
| AB (287 pg/uL) | BC (260 pg/uL) | CA (223 pg/uL) | |
| 5x Q5 Buffer | 30 uL
| ||
| 10 mM dNTPs | 3 uL
| ||
| 10 uM For (F-03) | 7.5 uL
| ||
| 10 uM Rev (G-03) | 7.5 uL
| ||
| Template (500 pg) | 5.22 uL | 5.76 uL | 6.73 uL |
| Q5 Polymerase | 1.5 uL
| ||
| 5x GC enhancer | 30 uL
| ||
| ddH2O | 65.28 uL | 64.74 uL | 63.77 uL |
| Total | 150 uL | ||
| 98 C | 30 sec |
| 98 C | 10 sec |
| 66 C | 15 sec |
| 72 C | 15 sec |
| repeat from step 2 | 30x |
| 72 C | 2 min |
| 12 C | hold |
4 Results for second PCR
∙ Visualized on 2% gel. Used 2 uL 50 bp ladder.
Figure 2: Results from large scale amplification of MaSp using post-elution
primers. The amplification is inconsistent.