For design considerations see Designing and Generating CRISPR-Cas Mutants

• 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.