inoue法制备超级感受态细胞-丁香通.docx

Inoue法制备大肠杆菌超级感受态细胞 关键词：Inoue法大肠杆菌 感受态2008-07-21 00:00 来源：互联网 点击次数：1704 实验步骤： 1、 Inoculate from an overnight grown in LB. 从培养过夜的 LB平板上挑取单菌落 。 2、 Grow in 250 ml SOB at 18 C until OD 6oo = 06(0.3)接种于 250ml SOB , 18 度培养至 OD = 0.6。 3、 On ice for 10 minutes. 菌液置冰上 10 分钟。 4、 Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge)for 10 min.at 4 C .4 度 2500g 离心 10 分钟。 5、 Resuspend cells gently in 80 ml of ice cold TB. 小心用 80ml 预冷 TB 重悬细胞。 6、 On ice for 10 minutes.(30min) 菌液置冰上 10 分钟。 7、 Spin at 2500 x g (5000 rpm in a Sorvall GSA,5500 rpm in a Sorvall SS-34,or 3000 rpm in a Beckman J-6B centrifuge)for 10 min.at 4 C .4 度 2500g 离心 10 分钟。 Resuspend cells gently in 20 ml of ice cold TB. 小心用 20ml 预冷 TB 重悬细胞。 9、 Add DMSO to a fin al co ncen tration of 7%. 加入 DMSO 至终浓度 7%。 10、 Place on ice for 10 minutes. 置冰上 10 分钟。 11、 Aliquot into 1-2 ml and freeze in liquid nitrogen. 分装，液氮速冻 。 12、 Store in liquid nitrogen. 冻存。 SOB Medium and TB (Tra nsformation Buffer) 2% (w/v) bacto trypt one 0.5% (w/v) yeast extract 10 mM Pipes 10 mM NaCl 55 mM MnC2 2.5 mM KCl SOB10 mM MgC 2.5 mM KCl SOB 10 mM MgC2 10 mM MgSO pH 6.7 - 7.0 TB 250 mM KCl 在加入 MnCb之前先用5NKOH调pH值到6.7 , adjust pH to 6.7 with 5N KOHprior to adding the MnCb tha nks to Markus Schn eema nn for the tip! Note: Compete nt cells are fragile (cell wall is thought to be weake ned),therefore treat the cells gently when preparing these cells.Do not vortex or pippette up and down to resuspend the cells.Do not spin the cells at too great a speed (sp inning dow n at 5000g will cause some cells to lyse). Always keep the cells chilled whe n maki ng compete nt cell.Do not let them warm up. Freez ing the cells appear to make cells more compete nt. Some cell strains may work better than others (DH5alpha works well in my hand).Note also that some cells (e.g.HB101)has greater recomb in atio n activity tha n others. This method does nt appear to work with BL21,so just grow the cells at 30 or 37 °C whe n maki ng BL21 compet