基因敲除详细步骤.docVIP

The following protocols take MLCK (myosin light chain kinase) as an example. General steps: BAC extraction (It is necessary for us to identify the BAC by PCR) Transform BAC to EL350 ( Cm+) Retrieving (Cm+ Amp+) 4. Targeting 1st loxP (Amp+ Amp+ and K+) 5. Transform MLCK 1st loxP to EL350 to get purify MLCK 1st loxP ( Amp+ and K+) 6. MLCK 1st loxP pop out (Amp+ and K+ AmP+) 7. Transform MLCK 1st loxP pop out to EL250 (Amp+) 8. Targeting 2nd loxP (Amp+ Amp+ and K+) 9. Transform MLCK 2nd loxP to DH-5α or XL1-Blue ( Amp+ and K+) 10. Linearization 1. BAC extraction So