Are DH5 alpha competent cells dam+ or dam-, and to be clear...if they are dam+ does that mean that they methylate DNA, and that restriction enzymes that are dam sensitive would NOT digest DNA isolated from DH5 alpha cells?
I'm trying to digest a plasmid isolated from DH5 alpha cells with BsaBI (NEB) but it's not cutting....
Thanks!
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#2
vladooo
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Posted 15 April 2010 - 11:29 AM
Yes, DH5a is dam+ (see http://openwetware.o..._coli_genotypes) and yes it blocks your BsaBI digestion. You have to subclone vector into dam- cells.
Edited by vladooo, 15 April 2010 - 11:31 AM.
#3
HomeBrew
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Posted 15 April 2010 - 04:58 PM
If you're in the US, NEB will send you E. coli K12 ER2925 (dam- , dcm-) for free with any order... Need some more BsaBI?
