Haynes Lab:Notebook/CRISPR Editing/2014/10/27: Difference between revisions
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:95°C 5 min | :95°C 5 min | ||
:ramp down to 25°C at 5°C/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 | |||
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Revision as of 14:45, 27 October 2014
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10/27/2014Phosphorylated and annealed gRNAs 23, 25, 31-38 for cloning into pX330.
Anneal in a thermocycler using the following parameters:
Incubate the ligation reaction in a thermocycler:
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