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文献和实验PCR引物设计和优化(PCR PRIMER DESIGN AND REACTION OPTIMISATION)
of amplification, as the mean length of target DNA is decreased: for templates of 300bp or less, denaturation temperature may be reduced to as low as 88℃ for 50% (G+C) templates (Yap and McGee, 1991), which means one may do as many as 40 cycles
of a 10% gel 4 ml H2O 3.3ml 30% acrylamide/bisacrylamide (29:1 mix) 2.5 ml1.5M Tris(PH 8.8) 10% SDS 100 µl 10% ammonium persulfate 100 µl TEMED 8 µl 2. Stacking gel: 4 ml 2.7ml water 1 ml 0.67ml 30% acrylamide/bisacrylamide (29:1 mix) 0.ml 1.0M
: a) to the 40 ul of digested cDNA add: 1 ul Taq I adaptor (45uM) 1 ul Nco I adaptor (5uM) 2.5 ul 10 mM ATP 0.5 ul NEB T4 DNA ligase buffer 0.2 ul T4 DNA ligase 45 ul total b) 37C for 3hrs or ON @ 30C
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