Team:UCLA/Notebook/Spider Silk Genetics/6 July 2015
7/6/2015
MaSp2 Sequencing Core Assembly
- PCA for MaSp2 Sequencing Core to be used for sequencing longer MaSp constructs.
- Used the MaSp2 Seq Core 1-F (D-05) and MaSp Seq Core 1-R (E-05) to create full length MaSp sequencing core.
| Volume (uL) | |
|---|---|
| 5x Q5 Buffer | 5 |
| 10 mM dNTPs | 0.5 |
| 10 uM For (D-05) | 1.25 |
| 10 uM Rev (E-05) | 1.25 |
| 5x GC Enhancer | 5 |
| ddH2O | 11.75 |
| Q5 Polymerase | 0.25 |
| Total | 25 |
- NEB Tm calculator gives 65 C for annealing, based on the overlap between the forward and reverse.
- Test 63 C and 66 C.
| 98 C | 30 sec |
| 98 C | 10 sec |
| 63, 66 C | 15 sec |
| 72 C | 10 sec |
| repeat from step 2 | 25 x |
| 72 C | 2 min |
| 12 C | hold |
Results
- Cast 1.5 % TAE gel, used 2 uL of 50 bp ladder to visualize.
- The assembly was successful.