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. 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.