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Richard Powell Department of Microbiology, National University of Ireland, Galway, Ireland Frank Gannon European Molecular Biology Labora

Subcloning should be easy and fast, and work every time. The following protocols minimize the number of manipulations required to prepare DN

(adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu) A. So

平端连接 通常情况下，PCR产物可直接与平端载体DNA进行连接，但其连接效率效低。因为TaqDNA聚合酶具有非模板依赖性末端转移酶活性，能在两6条 DNA链的3'末端加上一个多余的碱基，使合成的PCR产物成为3'突出一个碱基的DNA分子。这种DNA分子的连接效率很低。由于PCR

competent cells (DH5alpha at competency of 108 cfu/µl of pUC18). The transformation procedure it self is very similar to a standard CaCl2 tr

C. G. Sagerstr¨om,1 B. I. Sun,1 and H. L. Sive2 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,Massachusetts

This method is after Marchuk,D.,et al.,1991,Nucl.Acids Res.19(5),pp1154. You will need: 10 x Taq buffer (Promega) Taq Polymerase (Promega

Linker Ligation (with T4 ) DNA Ligase In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE b

The overall sequence of events is: • Titer and plate out phage • Lift plaques onto filters and prepare them for screening • Make a probe

I. Initial mixture 5 μl dd H2O 1 μl PCR product 1 μl ligation buffer 2 μl TA vector (add second to last) 1 μl ligase (add last) Incuba