Team:UCLA/Notebook/Spider Silk Genetics/27 May 2015
5/27/2015
1. VF/R Amplification of MaSp 2CA Temperature Test
- Due to lower yield of previous reactions, we decided to test lower temperatures which may give a higher yield.
- We tested amplification at 63 C, 60 C and 57 C in 25 uL reactions using the following protocol.
| 3x Reaction Volume (uL) | |
|---|---|
| 5x Q5 Buffer | 15 |
| 10 mM dNTPs | 1.5 |
| 10 uM For | 3.75 |
| 10 uM Rev | 3.75 |
| Template (110 ng/reaction) | 1.5 |
| 5x GC Enhancer | 15 |
| Q5 Polymerase | 0.75 |
| ddH2O | 33.75 |
| Total | 75 |
| 98 C | 30 sec |
| 98 C | 10 sec |
| 63/60/57 C | 15 sec |
| 72 C | 15 sec |
| repeat from step 2 | 18x |
| 72 C | 2 min |
| 12 C | hold |
- The reactions were prepared as a mastermix without the addition of polymerase. Reactions were split, then polymerase added indivdually.
2. Resutls
- Cast 150 mL of 1.5% TAE agarose gel. Used 2 uL of NEB 50 bp ladder.
- The intensity of the bands at each temperature are approximately equal.
- For this test, we used primers reconstituted from stock. Compared to previous amplifications (5/19/2015 and 5/20/2015) the yield of amplification is improved. The old primers were probably degraded.
- There is a smear around 50 bp which may be leftover primers.
- We will use 63 C as annealing for future amplifications.