On trying to amplify acsA-up+accBCDA+acsA-dn off of a ligation
- Been trying to do this for a week
- Latest problem: ligation appears to have correct size band (5.5kb), but amplification doesn't work
- Will do Phusion temperature gradient, since 5.5kb template may act differently than .5kb
- FTR the primers (acsA-UF, acsA-DR) should have the same melting temperature
- acsA-UF: GATTTTCAAGCCCAGGTGACTG
- acsA-DR: CTATATCTGGCAAACAACTTTGGC
| Temp
|
Time
|
| 95 |
2:00
|
| 95 |
:30
|
| 47-63 |
:30
|
| 72 |
1:40
|
| Repeat |
35x
|
| 72 |
4:00
|
| 4 |
Hold
|
| Reagent
|
In ea (25ul)
|
M. mix
|
| Phusion |
0.5 |
4.5
|
| dNTP |
0.5 |
4.5
|
| acsA-UF |
0.5 |
4.5
|
| acsA-DR |
0.5 |
4.5
|
| Ligation |
0.5 |
4.5
|
| HF buffer |
5 |
45
|
| Water |
17.5 |
157.5
|
- Ran 2 gels last week where the ladder looked like hell
- Maybe got left out? Nucleases got in?
- Running all ladders (2 & 4 ul 2-log) in iGEM lab out on 1% agarose gel, 90V 30 min
- RESULT: Ladders looked okay (and compared to Matt's upstairs)
- Some loss of definition of closer lines
- Previous issues must have arisen from autofocus seeing so much DNA in extraction lanes?
|
Project name
