Objective
Troubleshoot ligation (see notes from last week)
- transform old ligation into commercial cells
- re-ligate O/N @ 16°C
- start double-digest again
Prep media, if needed, for ER2566 competent cells (just make and use fresh once DNA is ready)
Bench work
- Double-digest
Tube |
sterile H2O |
10X NEB2 |
100X BSA |
DNA |
NheI (10 kU/mL) |
SapI (2 kU/mL
|
pTXB1His
|
34.5 μL |
5 μL |
0.5 μL |
10 μL of 102 μg/mL |
2.5μL |
2.5μL
|
pTXB1
|
34.5 μL |
5 μL |
0.5 μL |
10 μL of 82 μg/mL |
2.5μL |
2.5μL
|
BSA PCR
|
24.5 μL |
5 μL |
0.5 μL |
15 μL from purified PCR reaction |
2.5μL |
2.5μL
|
- → 2h @ 37°C
- → 30' @ 65°C
- → store insert reaction @ -20°C
- Phosphatase vectors
- add 1μL Alkaline Phosphatase to double-digest of pTXB1His and pTXB1 from step #1
- → 15' @ 37°C
- → store @ -20°C
- Transformation into NovaBlue
- 5μL pTXB1His+BSA ligation + 50μL cells
- 5μL pTXB1+BSA ligation + 50μL cells
- Followed standard transformation protocol for each sample in step #1
- → heat shock step was 45"
- → plated 100μL each on LBAmp and stored the rest @ 4°C
- Kathryn Muratore 15:49, 29 June 2011 (EDT): I forgot to do the O/N ligation @ 16°C!
|