遗传与分子生物学实验 (24).pdf

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1、Preparation of competent E.coli cells for transformationThis protocol is based on the Inoue Method(Molecular Cloning 3rd,Chapter 1,Protocol 24)with modifications.ReagentsReagents1.SOBDissolve the following components in 800 ml H2O,and then add H2O to 1 L.Autoclave to sterilize.ReagentFinal concentra

2、tionStock1 Lyeast extract0.5%/5 gtryptone2%/20 gNaCl10 mM2.5 M4 mlKCl2.5 mM1 M2.5 mlMgCl210 mM1 M10 mlMgSO410 mM1 M10 ml2.DMSO:prechill to 0C before use.3.TB(Transformation Buffer)(1)Prepare 0.5 M PIPES(pH 6.7)by dissolving 15.1 g of PIPES in 80 ml of H2O.Adjust the pH to 6.7 with 5 M KOH,and then a

3、dd H2O to 100 ml.Sterilize thesolution by filtration through a Nalgene filter(0.45 m pore size).Store at-20.(2)Dissolve CaCl22H2O,KCl and PIPES in 800 ml of H2O as listed below.Adjust the pH to 6.7 with KOH or HCl and then add 10.88 g of MnCl24H2O.AddH2O to 1 L.Sterilize by filtration with 0.45 m Na

4、lgene filter and store at 4.ProcedureProcedure1.Inoculate 20 l of E.coli glycerol stock(DH5,BL21，HT115 etc.)into 5 mlof LB.37 overnight.2.Use this overnight culture to inoculate three 1L flasks containing 250 ml ofSOB.The first flask receives 250 l(1/1000)of overnight culture,the secondone receives

5、500 l(1/500),and the third receives 1.25 ml(1/200).3.Incubate the cultures until one of them reaches D600=0.50.55.Use this culturefor the following steps and discard the two other cultures.*You may need to modify STEP 2 and STEP 3 as the strains and incubatingconditions vary.*In our lab,you can refe

6、r to the following incubating conditions:DH5:2.5 ml of overnight culture(1/100),20/200 rpm,21 h.BL21:250 l of overnight culture(1/1000),37/200 rpm,4 h.HT115:250 l of overnight culture(1/1000),37/200 rpm,4.5 h.4.Place the flask in ice for 10 min.*Cover the flask with ice as much as possible.5.Harvest

7、 the cells by Cfg 4000 rpm/4/10 min,in 50 ml centrifuge tubes.Discard the supernatant.6.Gently resuspend the pellet in each tube in 80 ml of ice-cold TB.Store on icefor 10 min.*DO it gently!Molecular Cloning recommends swirling for resuspention,whichis hard to do.Weve tried resuspend by pipetting ge

8、ntly,and it worked well.7.Cfg 4000 rpm/4/10 min.8.Gently resuspend the pellet in 20 ml of ice-cold TB and 1.4 ml of DMSO.*DMSO needs to be stored at-20before use.9.Aliquot into prechilled,sterile tubes.Immediately freeze the competent cellsby immersing the tightly closed tubes in a bath of liquid ni

9、trogen.10.Store at-70.Normally they can be used for years.11.Use 1ng and 5ng of pBluescript DAN to transform 100ul of competentcells.Count the colonies on the LB(with Amp)plates next day to calculate thetransformationefficiency.(1ngDNA normallygivesabout500-1000colonies.)KeyKey pointspoints1.Stage o

10、f the cells:OD600=0.50.55.2.Culture the cells at low temperature(like 20)will increase the efficiency.3.Always keep the cells on ice.4.Operating gently.5.Transfer competent cells to-70 after freezing by liquid nitrogen as soon aspossible.6.Prechill everything possible:(1)Centrifuge tubes(2)Eppendorf tubes or other tubes that is to store the competent cells.(3)Tips(4)Reagents:Transformation Buffer(ice-cold),DMSO(-20).