Haynes Lab:Notebook/CRISPR Editing/2014/10/29: Difference between revisions
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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==10/29/2014== | ==10/29/2014== | ||
Phosphorylated and annealed gRNAs 26, 30, 39-49 for cloning into pX330. | |||
{| {{table}} | |||
|- | |||
|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 | |||
<br> | |||
Dilute the annealed oligo 1:250 (250-fold) | |||
<br> | |||
Set up digestion-ligation reaction: | |||
{| {{table}} | |||
|- | |||
|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 | |||
<br> | |||
Long transformation, used plates made by cameron and my cells. <br>used 50ul of cells, didn't do positive transformation controls because these cells have been working really well<br>didn't spin after recovery, just plated 350ul of the transformed cells in SOC | |||
<br><br> | |||
specs for minipreps for gRNAs in pX330 23, 25, 31-38 | specs for minipreps for gRNAs in pX330 23, 25, 31-38 | ||
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| ||0.094||0.049||1.911||94.206 | | ||0.094||0.049||1.911||94.206 | ||
|} | |} | ||
<br><br> | |||
Repicked g033, g037, and g038 to grow up tonight, miniprep tomorrow morning. | |||
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Latest revision as of 00:29, 27 September 2017
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10/29/2014Phosphorylated and annealed gRNAs 26, 30, 39-49 for cloning into pX330.
Anneal in a thermocycler using the following parameters:
Incubate the ligation reaction in a thermocycler:
|