Haynes Lab:Notebook/CRISPR Editing/2015/05/25

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05/25/2015

Cloning gRNAs into pX330g
Anneal oligos for gRNAs

46
32
34
31
25
44
48
51
53
54
55
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 and then ramp down to 25°C at 5°C/min


Dilute the annealed oligo 1:250 (250-fold).

Set up digestion-ligation reaction:

Xul pX330 or other backbone vector (100ng)
2ul phosphorylated and annealed oligo duplex from step 1 (1:250 dilution)
2ul 10X Tango buffer (or FastDigest Buffer)
1ul DTT (10mM to a final concentration of 1mM)
1ul ATP (10mM to a final concentration of 1mM)
1ul 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°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

Transform 1-2ul final product into cells

Run qPCR products from plate on gel, find out what the band in the no template control looks like compared to the gDNA positive wells