10/29/2014
Phosphorylated and annealed gRNAs 26, 30, 39-49 for cloning into pX330.
| 1 ul oligo 1 (100µM)
|
| 1 ul oligo 2 (100µM)
|
| 1 ul 10X T4 Ligation Buffer (NEB)
|
| 6.5 ul ddH2O
|
| 0.5 ul T4 PNK (NEB)
|
| 10 ul total
|
Anneal in a thermocycler using the following parameters:
- 37°C 30 min
- 95°C 5 min
- ramp down to 25°C at 5°C/min
Dilute the annealed oligo 1:250 (250-fold)
Set up digestion-ligation reaction:
| X ul pX330 or other backbone vector (100ng)
|
| 2 ul phosphorylated and annealed oligo duplex from step 1 (1:250 dilution)
|
| 2 ul 10X Tango buffer (or FastDigest Buffer)
|
| 1 ul DTT (10mM to a final concentration of 1mM)
|
| 1 ul ATP (10mM to a final concentration of 1mM)
|
| 1 ul FastDigest BbsI (Thermo Fisher Fermentas)
|
| 0.5 ul T7 DNA ligase
|
| Y ul ddH2O
|
| 20 ul total
|
Incubate the ligation reaction in a thermocycler:
- 37 5°C 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, used plates made by cameron and my cells.
used 50ul of cells, didn't do positive transformation controls because these cells have been working really well
didn't spin after recovery, just plated 350ul of the transformed cells in SOC
specs for minipreps for gRNAs in pX330 23, 25, 31-38
| Sample Read#
|
260
|
280
|
260/280
|
ng/µL
|
| g023 |
0.14 |
0.074 |
1.897 |
140.269
|
| g025 |
0.166 |
0.088 |
1.9 |
166.412
|
| g031 |
0.158 |
0.083 |
1.904 |
157.698
|
| g032 |
0.136 |
0.072 |
1.885 |
135.981
|
| g033 |
0.008 |
0.004 |
1.945 |
8.34
|
|
0.009 |
0.004 |
2.044 |
8.645
|
| g034 |
0.112 |
0.059 |
1.901 |
111.632
|
| g035 |
0.113 |
0.06 |
1.892 |
113.472
|
| g036 |
0.147 |
0.079 |
1.864 |
146.554
|
| g037 |
0.015 |
0.008 |
1.866 |
14.866
|
|
0.017 |
0.009 |
1.859 |
16.959
|
| g038 |
0.084 |
0.044 |
1.894 |
84.224
|
|
0.094 |
0.049 |
1.911 |
94.206
|
Repicked g033, g037, and g038 to grow up tonight, miniprep tomorrow morning.
| 
Project name
