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 Solution I: Tris.Cl 0.025 M EDTA 0.01M Glucose 0.05M pH 8.0 Solution II: SDS 1 % NaOH 0.2M fresh prepared (1Volume 2% SDS + 1Volume 0.4M NaOH) Solution III: (120 ml 5 M KAc + 23 ml HAc + 57 ml H2O) / 200 ml Protocol: 1. Harvest 50 ml bacterial cells (O/N) by centrifugation at 9,000 r/m for 10 min, pour off the supernatant clearly. Add 5ml ice-cold Solution I, mix. Add 10 ml Solution II, invert several times gently. Add 7.5 ml Solution III, invert several times gently. 5. Centrifuge at 9,000 r/min for 10 min at 4℃. Remove the supernatant to a fresh tube. Add 0.6 volume of isopropanol Centrifuge at 9,000 r/min for 10 min at 4℃. Remove supernatant. Dissolve DNA pellet in 400 ul TE￥, add 20 ul 10mg/ml RNase, 55℃, 20-30min. Add equal volume of Phenol/chloroform (1:1), mix and centrifuge at 12,500 rpm for 5 min at RT. (From this step, 1.5 ml tube can be used) Transfer supernatant to a fresh tube, add equal volume of chloroform, mix and centrifuge at 12,500 rpm for 10min at RT Add 0.1 volume of 3M NaAc (pH5.3) and 2 volume of ethanol (stored at -20℃). Mix and centrifuge at 12,500 rpm for 10 min at 4℃. Dissolve DNA with 50 ul pH 7.4 MilliQ H2O. (TENS isn’t good for BAC extraction) 2. Transform BAC into EL350 (Cm+) Pick up a single colony of EL350 to 3ml LB￥, grow at 32℃ O/N (12-16h) Next day, incubate 1 ml of O/N culture