Inoue法制备超级感受态细胞-丁香通.docVIP

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 until OD600?= 0.6.(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 4.4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 4.4度2500g离心10分钟。 8、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 final concentration 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 (Transformation Buffer) SOB? 2% (w/v) bacto tryptone? TB? 0.5% (w/v) yeast extract? 10 mM?Pipes? 10 mM?NaCl? 55 mM?MnCl2? 2.5 mM?KCl? 15 mM?CaCl2? 10 mM?MgCl2? 250 mM?KCl? 10 mM?MgSO4? 在加入MnCl2之前先用5N KOH调pH值到6.7，adjust pH to 6.7 with 5N KOH prior to adding the MnCl2?thanks to Markus Schneemann for the tip!? pH 6.7 - 7.0? Note: Competent cells are fragile (cell wall is thought to be weakened),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 (spinning down at 5000g will cause some cells to lyse). Always keep the cells chilled when making competent cell.Do not let them warm up. Freezing the cells appear to make cells more competent. Some cell strains may work better than others (DH5alpha works well in my hand).Note also that some cells (e.g.HB101)has greater recombination activity than others. This method doesnt appear to work with BL21,so just grow the cells at 30 or 37 ℃ when making BL21 competent cells.However,it has been suggested that the efficien