Haynes Lab:Notebook/CRISPR Editing/2015/05/25: Difference between revisions

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==03//2015==
==05/25/2015==
'''Cloning gRNAs into pX330g'''<br>
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
<br>
Dilute the annealed oligo 1:250 (250-fold).
<br>
 
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
<br>
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
<br><br>
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
<br><br>




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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



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