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T/A-based subcloning of PCR amplified DNA fragments
Richard Powell Department of Microbiology, National University of Ireland, Galway, Ireland Frank Gannon European Molecular Biology Labora
2010-03-05
Easy Subcloning
Subcloning should be easy and fast, and work every time. The following protocols minimize the number of manipulations required to prepare DN
2010-03-05
Random subclone generation
(adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu) A. So
2010-03-05
PCR产物克隆方法
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
2010-03-05
CLONING FROM LOW MELT AGAROSE
competent cells (DH5alpha at competency of 108 cfu/µl of pUC18). The transformation procedure it self is very similar to a standard CaCl2 tr
2010-03-05
SUBTRACTIVE CLONING:Past, Present, and Future
C. G. Sagerstr¨om,1 B. I. Sun,1 and H. L. Sive2 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge,Massachusetts
2010-03-05
Construction of homemade 'T-vectors'
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
2010-03-05
Linker Ligation (with T4 ) DNA Ligase
Linker Ligation (with T4 ) DNA Ligase In a microcentrifuge tube prepare a solution of blunt ended, dephosphorylated DNA (100-500ng) in TE b
2010-03-05
Clone Genes From a Phage Library
The overall sequence of events is: • Titer and plate out phage • Lift plaques onto filters and prepare them for screening • Make a probe
2010-03-05
TA Cloning
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
2010-03-05
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