Haynes Lab:Notebook/CRISPR Editing/2014/10/29

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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
g0230.140.0741.897140.269
g0250.1660.0881.9166.412
g0310.1580.0831.904157.698
g0320.1360.0721.885135.981
g0330.0080.0041.9458.34
0.0090.0042.0448.645
g0340.1120.0591.901111.632
g0350.1130.061.892113.472
g0360.1470.0791.864146.554
g0370.0150.0081.86614.866
0.0170.0091.85916.959
g0380.0840.0441.89484.224
0.0940.0491.91194.206



Repicked g033, g037, and g038 to grow up tonight, miniprep tomorrow morning.


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