04/08/15
Assembly
- Calculations
- Vector - MV2(E/X); 50 ng = 1.5 μL
- Insert - BD004(E/S); (4096/4529)*2*50 = 90.4 ng = 2.8 μL
- BD004 + MV2
- MV2 only (neg)
- BD004 only (neg)- to check if any of old vector co-purified w/ insert; bands were very close on gel
Reagent
|
1
|
2
|
3
|
DNA insert |
2.8 |
--- |
2.8
|
DNA vector |
1.5 |
1.5 |
---
|
2x buffer |
5.0 |
5.0 |
5.0
|
T4 ligase |
0.5 |
0.5 |
0.5
|
dH2O |
0.2 |
3.0 |
1.7
|
|
10.0 μL |
10.0 μL |
10.0 μL
|
--> RT/ ~30 min.
- Add 50 μL DH5-α turbo
- 5 min./ ice
- Plate on 100 ug/mL amp agar. Grow at 37 °C o/n
RESULTS
- BD004 + MV2 = ~10 colonies
- MV2 only (neg) = 0 colonies
- BD004 only (neg) = ~15 colonies
- Possible success: Insert-only had more colonies than insert+vector. Pik 6 colonies for 5 mL cultures/ streak plate. Verify minipreps via restriction digests.
|