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文献和实验Single tube confirmation PCR protocol
Polymerase. - Add 35 µl of the PCR mix to each of the 20 PCR tubes that already contain the 15 µl of primers and template. 110 µl 5 µl 10 x Taq buffer(see below) 11 µl 0.5 µl 20 mM dNTP's (0.2 mM) 11 µl 0.5 µ
Purification of Plasmid from 50 ml-culture
1. Shake E. coli harboring plasmid at 37 C overnight in 50 ml of TB containing appropriate antibiotics. (when using ampicillin, addition of the antibiotics to 100-200 ug/ml rather than usual 50 ug/ml may improve the yield of plasmids.)
ES CELL DNA EXTRACTION: TUBE ME
in an eppendorf tube to recover more of the DNA . NOTE- THIS TAKES A LOT OF TIME if you do the whole plate this way! Lysis buffer: ( 100 ml Recipe ) 10 mM Tris-HCl pH7.5 - 0.5 ml of 2M 10 mM EDTA - 2 ml of 0.5M 10 mM NaCl - 0.2 ml
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