Team:UCLA/Notebook/Spider Silk Genetics/22 May 2015
iGEM UCLA
5/22/2015
1. Amplification of MaSp2 2CA
- We scaled up to 8x 50 uL reactions to amplify MaSp using the VF/R primer pair for conducting digestion and ligation testing.
- For the template, we used 2CA diluted 1:1000 for a final concentration of 219 pg/uL.
- We used the protocol below:
|
|
Volume (uL)
|
| 5x Q5 Buffer
|
81
|
| 10 mM dNTPs
|
8.1
|
| 10 uM For
|
22
|
| 10 uM Rev
|
22
|
| Template
|
8.1
|
| 5x GC buffer
|
81
|
| Q5 Polymerase
|
4
|
| ddH2O
|
4.95
|
| Total
|
410
|
| 98 C
|
30 sec
|
| 98 C
|
10 sec
|
| 66 C
|
15 sec
|
| 72 C
|
15 sec
|
| repeat from step 2
|
18x
|
| 72 C
|
2 min
|
| 12 C
|
hold
|
- The reaction was set up as a master-mix without polymerase, which was added after distribution into the strips.
2. Results
- Olivia PCR purified using two columns:
|
|
Concentration (ng/uL)
|
A 260/280
|
| 2CA-1
|
42.08
|
4.45
|
| 2CA-2
|
35.77
|
5.81
|
