Team:UCLA/Notebook/Spider Silk Genetics/20 May 2015
5/20/2015
1. VF/R Amplification of MaSp Plasmid
- Based on yesterday's results with amplifying 2CA using the VF/R primer pair, we decided to scale up to obtain enough plasmid to perform digestion tests.
- We used the following protocol, and set up 2x 50 uL for amplification of 2CA.
Amount (uL) | |
---|---|
5x Q5 Buffer | 10 |
10 mM dNTPs | 1 |
10 uM For | 2.5 |
10 uM Rev | 2.5 |
2CA (219 pg/ul) | 1 |
5x GC Buffer | 10 |
Q5 Polymerase | 0.5 |
ddH2O | 22.5 |
Total | 50 |
98 C | 30 sec |
98 C | 10 sec |
66 C | 15 sec |
72 C | 15 sec |
repeat from step 2 | 17x |
72 C | 2 min |
12 C |
- 2CA was diluted from 219 ng/uL 1:1000 in ddH2O to 219 pg/uL.
2. Results
- We visualized the results on 2% TAE agarose gel using 2 uL NEB 50 bp ladder.
- The presence of a smear under 50 bp may indicate incomplete PCR and leftover primers. We will try 20 cycles in the future to see if better amplification can be achieved.
- We excised the bands at ~450 bp for gel extraction tomorrow.