Cg13032Electrocompetent cells制备.docVIP

C. glu 13202 Electrocompetent cells preparation and electroporation制备和转化方法的选择 Electroporation efficiency comparison between A, 2TY and LBHIS medium. I. A medium protocol as Efficient markerless-temperature-sensitive plasmid-2011 1. -80oC stock streaked to LBG solid medium, 30oC, 24h. One colony to 3ml LBG medium, O/N (~16h) culture at 30oC, 220rpm. 2. 2ml to 80ml Epo medium, 18oC , 220rpm culture to OD610=~0.5. 18C水降温, *低温培养(约过夜) 3. 40ml each to two cold 50ml tubes, 4oC, 5000rpm, 5min, pellet. Suspend with 1ml 15% cold glycerol, then add to 40ml, pellet 4. Repeat step 3.（3-4次） 5. Combine the suspension, add to 40ml, pellet. Finally suspended in 0.4ml. 100ul aliqout. Use now or frozen at -80oC.（比例：100ml菌—10管感受态） 6. Add DNA to cells, tap gently to mix, transfer to cold 0.1cm cuvette. Gene pulser 1.95 kV/cm and 200 Ω electroporation. 7. 1ml A medium added immediately, suspension immediately incubated for 6 min at 46oC in a water bath. *热休克 8. After 2 h recovery 200rpm at 25oC, cells were spread onto selective A medium, 30oC, 1-2 days. (2xTY+2xTY+A+A+A) 五种培养基：平板培养－液体试管－液体扩培－悬浮－平板筛选 II. 2TY, same procedure as above except growth media are all 2TY and outgrowth medium is SOC (2xTY+2xTY+2xTY-+2xTY+SOC) III. LBHIS medium, as Construction of a novel sacB-based-2012 [Jiangnan U]LBG for seed growth, Modified Epo media for large scale culture, LBHIS for suspension and selection. LBG media (LB media supplemented with 5 g/L glucose) Modified Epo media.10 g/L trypton, 5 g/L yeast extract, 10 g/L NaCl, 30 g/L glycine and 0.1% Tween 80. LBHIS.5 g/L trypton, 5 g/L NaCl, 2.5 g/L yeast extract, 18.5 g/L Brain Heart Infusion powder and 91 g/L sorbitol. 1.5% agar. (LBG+LBG+Epo+LBHIS+LBHIS) sorbitol [Sangon SB0491, 500g/54]. Brain Heart Infusion powder, Fluka 53283 [Sigma-Aldrich], 500G/696.15 2xTY for rountine culture: Dissolve16g Tryptone, 10g Yeast Extract, 5g NaCl in ~900ml of dH2O. Adjust pH to 7.0 with 5N NaOH. Add dH2O to 1L. Autoclave. For solid medium, add 1.5% agar. A medium (=