摘要:After an aliquot of the PCR mixture is analyzed on an agarose gel, the remainder of the reaction is concentrated by ethanol precipitation, resuspended in buffer, and subjected to a simultaneous fill-i[阅读全文]

摘要:This is a modification of the procedure in Short Protocols (1-41,1-45)1.Prepare a 50 ml liquid lysate:A.mix 2xlO8 E.coli cells with 100μl phage (from one picked plaque in 500μl SM),and 100μl [阅读全文]

摘要:After an aliquot of the PCR mixture is analyzed on an agarose gel, the remainder of the reaction is concentrated by ethanol precipitation, resuspended in buffer, and subjected to a simultaneous fill-i[阅读全文]

摘要:SummaryWCE Extracts are prepared from strain SHY282 (TFC4-Flag-EE (HpaI)).TFIIIC complex is first purified by flag affinity: TFC4 contains a single copy of the flag epitope.The flag purified TFIIIC is[阅读全文]

摘要:Purification of TFIIA from E.coliLinda Warfield,Hahn Lab,2003Volumes given are for 2L of cells for each subunit (Toa1 and Toa2).Toa1 and Toa2 subunits are expressed separately in cultures of OD0.6 for[阅读全文]

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摘要:Screen of GST-Fusion Protein Expression(pGEX system by Amersham: for check clones for expression of the desired fusion protein prior to large-scale purification)Pick several colonies of E.coli transfo[阅读全文]

摘要:Small scale fusion protein preparationGrow 5ml culture o/n in TB with amp.Add o/n culture to 50ml of TB amp and grow for 3 hours in 37℃ shaker.Induce culture by adding 20μl of 1M IPTG (final [0.4mM[阅读全文]

摘要:Making GST fusion proteins:(07/19/03)ver.1Grow up 5ml LB with Amp o/n.Add to 45ml LB with Amp37o shake 2.5 - 3 hrs,till OD600 0.4-0.8Put bottles in room temperature water for 10 min to cool down.Add 1[阅读全文]

摘要:GST Protein Prep.Ver.21)Grow 50ml of culture in LB or TB antibiotic o/n at 37℃ shaker.2)Dilute culture in LB or TB antibiotic 1:103)Grow 3hrs at 37℃.4)Induce culture by adding 0.4 mM IPTG final concen[阅读全文]

摘要:Low (“Low”)Imidazole Buffer 0.5L100mM Imidizole 3.4g5% glycerol 25ml 100% glycerol50mM Tris-HCl (pH 7.9)50ml 0.5M Tris-HCl (pH 7.9)0.1% Tween-20 0.5M 100% Tween-20500mM NaCl50ml 5M NaCldH2[阅读全文]

摘要:Inoculate 2ml of 5ml o/n culture of either p3133 (empty vector pET11a)or p3134 (dnEBNA-1/Soft)in E.coli BL21 LysS per 0.5L LB ampicillin (grow two 0.5L cultures of each)Incubate ~2hrs 37℃250rpm until [阅读全文]

摘要:J.Movius,K,Coachman,and S.Hahn (Hahn Lab)last modified 10/9/00Induction of BRF in bacteria and purification on Ni-NTA agaroseTransform BRF plasmid into strain BL21 DE3 (pLysS)or JM25 (DE3 containing t[阅读全文]

摘要:1. Materials(1) Antibody 7E3, 2L sup grown in flasks, frozen and thawed overnight. (2) BioRad Affi-Gel Protein A MAPS II Buffers cat. #1530-6160 ($161.00) (3) 50 mM Tris pH7.8.(4) 40 g ammonium sulfat[阅读全文]

摘要:1、Required Materials and Equipment(1) Protein A or G agarose gel column (10 ml or 5 ml of packed beads; see guidelines below for choice of protein A or protein G)(2) Ice-cold Tris-buffered saline (TBS[阅读全文]

摘要:Contributor: Suprya JayadevDate: Mar. 11, 1991I. Lower, chloroform phase:1) Dry on rotovapor system with house vacuum lines. It is not necessary to dry sample completely, but sufficiently to yield a f[阅读全文]

摘要:1. Purify plasmid from bacteria. We recommend the Qiagen EndoFree Plasmid Maxi kit for the purification of the targeting vector plasmid from bacteria. Please follow the directions in the kit. Electrop[阅读全文]