View source for Cloning CRISPR-Cas Plasmids

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[[ Category: Cloning ]]
[[ Category: Molecular Biology ]]
[[ Category: Genome Editing ]]
[[ Category: CRISPR ]]


For design considerations see [[ Designing and Generating CRISPR-Cas Mutants ]]

=Cloning=
* First digest vector by adding to a PCR tube (or use a frozen, pre-digested vector):
** 2 ug of pX335/pX459
** 1uL of 10X NEB Buffer 2.1
** 1 uL of CIAP
** 1 uL of BbsI
** water up to 10 uL
** Digest for 1h in, gel purify the fragment from an agarose gel, using the QIAEX II Kit and check the concentration by nanodrop.
* Next prepare the insert by annealing and phosphorylating the primers in a PCR tube:
** 4.5 uL of water
** 1 uL of a 100 uM stock of each oligo
** 2 uL of 5X ligase buffer
** 1 uL of T4 PNK
** Incubate at 37C for 30 mins then 95C for 5 mins to heat inactivate PNK then ramp down to 25C at 5C/min to allow the oligos to anneal, leave at room temperature
** Dilute the annealed oligos 250X in water (2 uL + 498 uL of water)
* Combine the ligation mixture in an eppendorf tube:
** 50 ng of vector 
** 3 uL of annealed insert or '''water as a blank'''
** 2 uL of 5X ligation buffer
** 1 uL of T4 DNA Ligase
** Water to 10 uL
* Incubate for 10 min at RT
* Transform 2uL of this into competent cells (see [[Transformation of Bacteria]]), plating the entire transformation.

===Verification===
* Miniprep the transformed clones and digest 1 ug of purified plasmid for 1h with AgeI and BbsI in NEB Buffer 1.1 and then run on an agarose gel.  Empty vectors will yield a ~1kb fragment, wheras vectors with insert will only be cut once.  This is because the cloning removes the BbsI site.
* Send clones with insert for sequencing with the hU6 sequencing primer. Go to http://bridgeslab.sph.umich.edu/protocols/index.php/Submitting_Plasmids_for_Sequencing for further details.  Align those clones with the empty vector full sequence or the hU6 sequencing of the empty vector, available at http://www.addgene.org/42335/sequences/.  Doublecheck that your insert is correct, and is oriented in the correct manner.