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. Linearization1. BAC extractionSolution I:Tris.Cl 0.025 MEDTA 0.01MGlucose 0.05MpH 8.0Solution II:SDS 1 %NaOH 0.2Mfresh prepared (1Volume 2% SDS + 1Volume 0.4M NaOH)Solution III:(120 ml 5 M KAc + 23 ml HAc + 57 ml H2O) / 200 mlProtocol: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 freshtube.6.Add 0.6 volume of isopropanol7.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 12,500rpm 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 andcentrifuge at 12,500 rpm for 10min at RT11.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 min4.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 pH7.4 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 construction1.PCR amplify two 500 bp homologous arms（BAC as template）2.Insert these two fragments to T vector3.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 colones5.Cut the retrieval plasmid with Hind III, purify the product.Transformation1.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=0.53.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 min5.Spin at 3,500 rpm for 6 min, resuspend cells with 1.5ml pH 7.4 MilliQ6.Spin, wash twice more.7.Remove supernatant, add about 50 μl pH 7.4 MilliQ8.Add 200-500ng linear retrieval plasmid to 50ul BAC-EL350, pipette them to anelectroporation cup (0.1 cM gap).9. 1.75kV, 25 uF, 200 ohms.10. Add 1ml SOC to each curvette and incubate at 32℃ for 1h.11. Centrifuge at 3,500 rpm for 3 min, pour off most of the supernatant and plate cells (Amp+).MLCK-PL2534. Targeting 1st lox P(Amp+ Amp+ and K+)￥sequenceMini-targeting vector construction1.PCR amplify 80 bp homologous arms lox P floxed Neo cassette (1st lox P) fromPL452.2.Ligate 1st lox P with T vector (If the PCR product quantity is enough for later use ,this procedure is not necessary)3.Cut 1st lox P–T to get 1st lox P fragment, purify the product.Note: If you use the ligation method to construct the mini-targeting vector, two ~500bp homologous arms are ligated to the floxed Neo cassette excised from PL452 with Eco RI and Bam HI and to PL253 that is linearized by Not I and Sal I.For more detail, pls see, Lee, E. C. et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics73,56-65 (2001)Transformation1.Pick up a single colony of MLCK-PL253-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=0.53.Induce MLCK-PL253-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 min5.Spin at 3500 rpm 6 min, resuspend cell with 1.5ml pH 7.4MilliQ6.Spin ,wash twice more.7.Remove supernatant, add about 50μl pH 7.4MilliQ8.Add 200ng 1st lox P fragment to 50ul MLCK-PL253-EL350, pipette them to anelectroporation cup (0.1 cM gap).9. 1.75kV, 25 uF, 200 ohms.10. Add 1ml SOC to each curvette and incubate at 32℃ for 1h.11. Centrifuge at 3,500 rpm for 3 min, pour off most of the supernatant and plate cells (Amp+ and K+).$Kanamycin concentration?HindIII EcoRcoR I5. Transform MLCK 1st lox P to EL350 to get purified MLCK 1st lox P ( Amp+ and K+) $??Note: EL350 contains multiple copies of plasmids, so there will be both recombinant and unrecombinant plasmids in EL350.6. 1st lox P pop out (Amp+ and K+ Amp+)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 2h to OD600≈0.4.3.Induce EL350 with 1mg/ml Arabinose at 32 ℃ for 1h.From this step, all on ice or in 4℃4.Transfer 6ml cells to 15 ml centrifuge tube, put on ice for 15 min5.Spin at 3500 rpm for 6 min, resuspend cells with 1.5ml pH 7.4 MilliQ6.Spin ,wash twice more7.Remove supernatant, add 50 μl pH 7.4 MilliQ8. Add MLCK 1st lox P plasmid (dilution≈1:1000) to 50μl competent cells, pipettethem to an electroporation cup (0.1 cM gap).9. 1.75kV, 25μF, 200 ohms.10. Add 1ml SOC to each curvette and incubate at 32℃for 1h.11. Centrifuge at 3,500 rpm for 3 min, pour off most of the supernatant, plate cells (Amp+).Note: You can plate cells to the K+ plate to conform the pop out efficiency (No or little electroporated cells grow on the K+ plate)HindIII EcoRHindIII EcoR I7. Transform MLCK 1st lox P pop out to EL250 (Amp+)Note: There may be leaky expression of the Cre recombinase in EL350, leading to undesired excision between floxed lox P sites in the final construct.8. Targeting 2nd lox P(Amp+ Amp+ and K+)Mini-targeting vector construction1.PCR amplify 80 bp homologous arms FRT-Neo-FRT-loxP cassette (2nd lox P)from PL451.2.Ligate 2nd lox P with T vector (If the PCR product quantity is enough for later use ,this procedure is not necessary)3.Cut 1st lox P–T to get 1st lox P fragment, purify the product.Note: If you use the ligation method to construct the mini-targeting vector, two ~500bp homologous arms are ligated to the FRT-Neo-FRT-loxP cassette excised from PL451 with Eco RI and Bam HI and to PL253 that is linearized by Not I and Sal I. Transformation1.Pick up a single colony of MLCK 1st lox P pop out EL250 to 3ml LB, grow at32℃ O/N (12-16h)2.Next day, incubate 1 ml of O/N culture to 50 ml LB, 32 ℃ for 2-3h to OD600=0.53.Induce MLCK 1st lox P pop out EL250 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 min5.Spin at 3,500 rpm 6 min, resuspend cell with 1.5ml pH 7.4MilliQ6.Spin ,wash twice more.7.Remove supernatant, add 50μl pH 7.4 MilliQ8.Add 200 ng 2nd lox p fragment to 50μl MLCK 1st lox P pop out EL250, pipettethem to an electroporation cup (0.1 cM gap).9. 1.75kV , 25μF , 200 ohms.10. Add 1ml SOC to each curvette and incubate at 32℃for 1h..11. Centrifuge at 3,500 rpm for 3 min, pour off most of the supernatant and plate cells (Amp+ and K+)HindIII EcoRHindIII EcoR9. Transform MLCK 2nd lox P to DH-5α or XL1-Blue ( Amp+ and K+) to purify the targeting vectorIt is important to confirm the restriction enzyme pattern.10. LinearizationSouthern Blot Protocol1.Following electrophoresis, cut the agarose gel to size by removing any blank oruntreated lanes that do not need to be transferred.2.Soak the gel in base solution for 2×20 minutes. There is no need to be treatedwith neutralizing solution.3.Allow genomic DNA to transfer overnight in base solution.4.UV crosslink, 125mJ/cm2. Then bake at 80℃ for 30 minutes.5.Hybridize the membranes with a labeled probe.Radioactive labeling of probe(Rediprime II Random Prime Labelling System, Amersham Biosciences)1.Dilute the DNA to be labeled to a concentration of 25ng in 45ul of TE buffer.2.Denature the DNA sample by heating to 100℃ for 5 minutes in a boiling waterbath.3.Snap cool the DNA by placing on ice for 5 minutes after denaturation.4.Add the denatured DNA to the reaction tube.5.Add 5ul of Redivue [32P] dCTP and mix by pepetting up and down about 12 times,moving the pipette tip around in the solution.6.Incubate at 37℃ for 1 hour, then room temperature overnight.7.Add 125ul EtOH and 5ul NaAc, centrifuge at 10 000 rpm for 10 minutes toprecipitate DNA. Dissolve DNA in 50ul dH2O.8.For use in hybridization, denature the labeled DNA by heating to 100℃for 5minutes, then snap cool on ice for 5 minutes. (without snap cooling, add hot probe into hybridization buffer, it works okey.)HybridizationInsert membrane in hybridization tube and wet with 65℃hybridization buffer. Increase volume slightly to adequately cover the membrane (approximately 5ml). Prehybridize at 65℃for 30~45 minutes. Replace the solution with freshhybridization buffer, and heat-denatured probe. Hybridize overnight while gently rotating at 65℃.Post hybridization treatmentWash the membrane in ~50ml of 2×SSPE, 0.1% SDS, 1mM EDTA (these is no difference whether the wash buffer is at 65℃ or room temperature) for 2×15 minutes. Wash twice for 15 minutes per wash in 0.2×SSPE, 0.1% SDS, 1mM EDTA at 65℃.RecipesBase Solution (1.5M NaCl, 0.5M NaOH)For 2 liters:175.35g NaCl40g NaOHQS to 2 liters with water and autoclave.20×SSPE (3M NaCl, 0.2 M NaH2PO4, 20mM EDTA, pH7.0)350.7g NaCl55.2g NaH2PO4(H2O) (or 48g anhydrous NaH2PO4)14.89g EDTADissolve in boiling water, cool to room temperature, and adjust pH to 7.0 with NaOH. QS to 2 liters with water and autoclave.2×SSPE 0.1% SDS, 1mm EDTA100 ml of 20×SSPE10 ml of 10% SDS2 ml of 0.5M EDTAQS to 1 liter with distilled water, skip autoclaving.0.2×SSPE 0.1% SDS, 1mm EDTA10 ml of 20×SSPE10 ml of 10% SDS2 ml of 0.5M EDTAQS to 1 liter with distilled water.Prepared byPeng Yajing & He Weiqi。