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文献和实验Screening lambda cDNA or genomic libraries
,and this will increase the efficiency of labelling considerably. - Boil the DNA to denature it for a few minutes.Briefly spin the tube and put on ice to cool.Add 6.6 µl 5X OLB,5 µl hot dATP (50 µCi),and 2 µl Klenow. - Allow the labelling reaction to proceed at room
Recombineering/Lambda red-mediated gene replacement
Electroprate linear DNA into electrocompetent cells Grow at 37°C on chloramphenicol plates PCR verify the deletion with oligos C and D (see below for design) Detailed procedure Day 0: Start overnight culture
糖凝胶分离的范围 试剂: (1)1 X TAE电泳缓冲液:45mmol/Ltris-硼酸,1mmol/LEDTA,pH8.0 (2)凝胶加样缓冲液:0.25%溴酚蓝,40%蔗糖水溶液。 (3)λ-DNA和提取的DNA λ-DNA是λ噬菌体的基因组DNA,全长50kb。用HindIII或(和)BamHI酶切得到的片段被广泛用于DNA电泳的分子量标准。 (4)分子量标准(&lambda
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