基因敲除具体步骤

The following protocols take MLCK (myosin light chain kinase) as an example.

General steps:

1.BAC extraction (It is necessary for us to identify the BAC by PCR)

2.Transform BAC to EL350 ( Cm+)

3.Retrieving (Cm+ Amp+)

4. Targeting 1st lox P (Amp+ Amp+ and K+)

5. Transform MLCK 1st lox P to EL350 to get purify MLCK 1st lox P ( Amp+ and K+)

6. MLCK 1st lox P pop out (Amp+ and K+ AmP+)

7. Transform MLCK 1st lox P pop out to EL250 (Amp+)

8. Targeting 2nd lox P (Amp+ Amp+ and K+)

9. Transform MLCK 2nd lox P 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.

2.Add 5ml ice-cold Solution I, mix.

3.Add 10 ml Solution II, invert several times gently.

4.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.

6.Add 0.6 volume of isopropanol

7.Centrifuge at 9,000 r/min for 10 min at 4℃. Remove supernatant.

8.Dissolve DNA pellet in 400 ul TE￥, add 20 ul 10mg/ml RNase, 55℃,20-

30min.

9.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)

10.Transfer supernatant to a fresh tube, add equal volume of chloroform, mix and

centrifuge at 12,500 rpm for 10min at RT

11.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℃.

12.Dissolve DNA with 50 ul pH 7.4 MilliQ H2O.

(TENS isn’t good for BAC extraction)

2. Transform BAC into EL350 (Cm+)

1.Pick up a single colony of EL350 to 3ml LB￥, grow at 32℃ O/N (12-16h)

2.Next day, incubate 1 ml of O/N culture to 50 ml LB, 32℃ for 2-3h to OD600=0.5 From this step, all on ice or in 4℃

3.Transfer 6ml cells to 15 ml centrifuge tube, put on ice for 15 min

4.Spin at 3,500 rpm for 6 min, resuspend cell with 1.5ml pH 7.4 MilliQ H2O.

5.Spin, wash twice more.￥

6.Remove supernatant, add about 50 ul pH

7.4 MilliQ H2O.

7. Add 10 ul BAC to 50 ul competent cells, pipette them to an electroporation cup (0.1 cM gap).

8. 1.75kV, 25 uF, 200 ohms.

9. Add 1ml SOC to each curvette￥and incubate at 32℃ for 1h.

10. Centrifuge at 3,500 rpm for 3 min, pour off most of the supernant and plate cells (Cm+).

3. Retrieving (Cm+ Amp+)

Retrieval plasmid construction

1.PCR amplify two 500 bp homologous arms（BAC as template）

2.Insert these two fragments to T vector

3.Not I, Hind III cutting and Spe I Hind III cutting 500 bp fragments ligated with Not I ,

Spe I cutting PL253￥

4.Transform, identify the positive colones

5.Cut the retrieval plasmid with Hind III, purify the product.

Transformation

1.Pick up a single colony of BAC-EL350 to 3ml LB, grow at 32℃ O/N (12-16h)

2.Next day, incubate 1 ml of O/N culture to 50 ml LB, 32 ℃ for 2-3h to OD600=0.5

3.Induce BAC-EL350 in 42℃ water bath by shaking for 15 min.

From this step, all on ice or in 4℃

4.Transfer 6ml cells to 15 ml centrifuge tube, put on ice for 15 min

5.Spin at 3,500 rpm for 6 min, resuspend cells with 1.5ml pH 7.4 MilliQ