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
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.