04/12/13
- Chem. Competent cell prep: CaCl wash; 15% glycerol/CaCl resuspension (used 30 mL instead of 20 mL); incubate in cold room on ice o/n
- MV9 building: colony PCR
MV9 building: colony PCR
- Pick 8 colonies from plates 1, 2, 3, and 4; make a mini-streak library, then wash pipette tip in PCR master mix
- Pick 4 colonies from plate 5 (neg. ctrl); do not add to streak library; include in PCR as neg ctrl.s
- Primers for MV2 vector: DD122B (fwd), DD123B (rev); see BBa_J176122
Reagent
|
Volume
|
x38
|
Expected: MV2 (empty) = 177 MV9 (single insert) = 253 extra inserts = +76
|
10 μL/lane, 1% agarose; Ladder
|
DNA (colony) |
--- |
---
|
10 μM primer 1 |
1.0 |
38.0
|
10 μM primer 2 |
1.0 |
38.0
|
2x GoTaq green |
10.0 |
380.0
|
dH2O |
8.0 |
304.0
|
|
20 μL |
|
Thermal cycling
- 95°C, 3 min.
- [95°C, 15 sec; 57°C, 15 sec; 72°C, 15 sec] x30
- 72°C, 3 min.
- 4°C, ∞
|