Team:UCLA/Notebook/Spider Silk Genetics/13 April 2015
4/13/2015
1 Polymerase Chain Assembly
The primers corresponding to the new MaSp2 sequences with a valine added to the end came today. We conducted polymerase chain assembly to place the biobrick prefix/suffix and the BsaI restriction enzyme recognition sites with the designed sticky ends.
We used NEB’s Q5 PCR annealing temperature calculator to determine that an annealing temperature of 65 C was optimal. We also decided to test annealing at 61.4 C and 59 C. In addition, we planned to use the GC enhancer because our sequence is GC-rich. We prepared reactions as follows:
| 1x (uL) | 3x (uL) | |
| 5x Q5 Buffer | 5 uL | 15 uL |
| 10 mM dNTPs | 0.5 uL | 1.5 uL |
| 10 uM For | 1.25 uL | 3.75 uL |
| 10 uM Rev | 1.25 uL | 3.75 uL |
| MaSp core (2.7 pg/uL) | 3.7 uL | 11.1 uL |
| 5x GC enhancer | 5 uL | 15 uL |
| Q5 Polymerase | 0.25 uL | 0.75 uL |
| ddH2O | 8.05 uL | 24.15 uL |
| Total | 25 uL | 75 uL |
| 98 C | 30 s | |
| 98 C | 10 s | |
| 65/61.4/59 C | 15 s | |
| 72 C | 15 s | |
| Repeat from step 2 | 20 x | |
| 72 C | 2 min | |
| 12 C | hold |
2 Results
We cast a 1.5% TAE gel to visualize the results. We used 2 uL of NEB 50 bp ladder.
Our results indicate that 65 degrees is too high, and does not have good yield, but 61.4 is too low and has non-specific amplification as shown by the smears. The band at 50 bp is attributed to primer dimer formation.
Based on our results, tomorrow, we will perform polymerase chain assembly with annealing at 64 C and with 25 cycles.