07/09/2014
Had about the same number of colonies on each of the ligation plates as the backbone. Will try the ligation following the Zhang lab protocol.
Will run through the protocol with pX330 and three gRNAs, g5, g13, g14
Annealing the oligos
oligo 1 (100μM)
|
1
|
oligo 2 (100μM) |
1
|
10X T4 Ligation Buffer (NEB) |
1
|
ddH2O |
6.5
|
T4 PNK (NEB) |
0.5
|
- 37°C 30 min
- 95°C 5 min
- ramp down to 25°C at 5°C/min
Ligation reaction
pX330 (185ng/ul)
|
0.540540541
|
1:250 annealed oligos |
2
|
10X Fast Digest buffer |
2
|
10mM DTT |
1
|
10mM ATP |
1
|
BbsI |
1
|
T7 DNA ligase |
0.5
|
water |
11.95945946
|
Thermocycler
- 37°C 5 min
- 23°C 5 min
- Cycle the previous two steps for 6 cycles (total run time 1h)
- 4°C hold until ready to proceed
Long transformation, 75ul cells
|