Haynes Lab:Notebook/CRISPR Editing/2014/10/27

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Phosphorylated and annealed gRNAs 23, 25, 31-38 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.