Designing and Generating CRISPR-Cas Mutants: Difference between revisions

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 ===Cloning===
-* First digest pX335 vector by adding to a PCR tube:
+* First digest pX335 vector by adding to a PCR tube (or use a frozen, pre-digested vector):
-** 1 ug of pX335,
+** 2 ug of pX335
 ** 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.
+** 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:
-** 1 uL of a 100 uM stock of each oligo with 4.5 uL of water
+** 4.5 uL of water
-** 2.5 uL of ligase buffer
+** 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
+** 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:
-** 5 ng of vector (~10 pmoles of vector)
+** 50 ng of vector
-** 3 uL of annealed insert (~30 pmoles of Insert) or water as a blank
+** 3 uL of annealed insert or '''water as a blank'''
-** 2.5 uL of 4X ligation buffer
+** 2 uL of 5X ligation buffer
 ** 1 uL of T4 DNA Ligase
 ** Water to 10 uL
 * Incubate for 10 min at RT
-* Transform into competent cells (see [[Transformation of Bacteria]])
+* Transform 2uL of this into competent cells (see [[Transformation of Bacteria]]), plating the entire transformation.