Team:UCLA/Notebook/Spider Silk Genetics/17 June 2015
iGEM UCLA
6/17/2015
PCR Amplifcation
- Set up 17x 25 uL reactions for each species of MaSp.
- Used 110 pg per 25 uL reaction
|
|
AB (112 pg/uL)
|
BC (205 pg/uL)
|
CA(219 pg/uL)
|
| 5x Q5 Buffer
|
85 uL
|
| 10 mM dNTPs
|
8.5 uL
|
| 10 uM For
|
21.25 uL
|
| 10 uM Rev
|
21.25 uL
|
| Template
|
16.70 uL
|
9.12 uL
|
8.54 uL
|
| Q5 Polymerase
|
4.25 uL
|
| 5x GC Enhancer
|
85 uL
|
| ddH2O
|
183.05 uL
|
190.63 uL
|
191.21 uL
|
| Total
|
425 uL
|
| 98 C
|
30 sec
|
| 98 C
|
10 sec
|
| 63 C
|
15 sec
|
| 72 C
|
15 sec
|
| repeat from step 2
|
18x
|
| 72 C
|
2 min
|
| 12 C
|
hold
|
PCR Purification
- We PCR purified using Zymo DNA Clean and Concentrator -5.
- Pooled 4x 50 uL reactions into one column.
- Right before elution with ddH2O, noticed some precipitate in the columns. Probably salts. We avoided touching it as much as possible.
Results
|
|
Concentration (ng/uL)
|
A 260/280
|
| AB
|
393.13
|
1.76
|
| BC
|
149.29
|
3.90
|
| CA
|
322.87
|
1.58
|
- AB appears fine
- The graph for BC was very messy, and there is probably error in the DNA concentration
- CA appears okay, but with some messy graph peaks.
- In the future, may want to use less template if this is a problem, and use fresh DNA and dNTPs
