Template:ZFN Enrichment Assay Protocol

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  1. Transform [PLASMID WITH zf+ , fokI, zf-, and w/out self-lysis device] into ? cells.
  2. Plate on whatever antibiotic the device is resistant to.
  3. Mix 1 uL Pbca9145-sbb23 PCR Product + 1 uL Pbca9145-1144#5 (both plasmid concentrations should be 1ng/uL, or diluted ~20x).
  4. Transform plasmid mixture (< 1 ng/ml) into cells with ZFN.
  5. Repeat the above 2 steps, transforming into cells without ZFN but with same ab resistance genes.
  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water to the cells
  3. Add 30 uL of KCM to the cells 
  4. Put your mixture on ice, let cool a minute or two
  5. Add 70 uL of the cell cocktail to the ligation, stir to mix
  6. Let sit on ice for 10 min
  7. Heat shock for 90 seconds at 42 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (Rescue)
  1. Add ?? uL arabinose.
  2. Plate overnight 70+ uL on ampicillin.