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Debbie

DH10B vs. DH5alpha

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Hi, Is there any situation where one would want to use DH10B over DH5alpha competent cells? I've read that DH10B is used for methylated DNA but I'm not sure what that means.

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You already got the right answer. To make it short:

bacteria possess certain restriction enzymes that degrade external DNA coming into the cell. In order to avoid restriction of own DNA they methylate it with a specific pattern that is recognized by the restriction system (which sometimes is part of the methlyation system) and restriction is avoided. DH5alpha has one major mutation in one of the restriction systems (hsdR, I think), that methylates (together with other subunits) own DNA and degrades unmethylated one. If you introduce unmethylated DNA in it, it won't get degraded.

DH10B got some more mutations in restriction systems (mcr-system, if memory serves) that can regognize methylated foreign DNA and degrades it. Thus in DH10B you can also insert DNA that was methylated (as you stated).

However, there is also DH5alpha MCR, that carries the same mutation, so it can also be used for methylated DNA.

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Hi

 

Im using DH10B and I wonder if they are able to methylate transfected DNA. I am asking because I am trying to cut with a methylation sensitive restriction enzyme (XbaI) that's cut site overlaps a known methylation site, but I don't know if this site will have been methylated by the DH10B strain. Thanks

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Hello,

 

you may have a problem there. The only methylation system (E. coli has several) that interferes with XbaI is Dam. Unfortunately I belive that DH10b are dam+. So if you isolate DNA out of DH10b chances are that it won't cut. So if you are dependent on that particular restriction site you can either

transform a dam- E. coli strain with the vector and then re-isolate it from it. Alternatively you can try to look for an isoschizomer that is not methylation-sensitive. I do not know one out of my head, though.

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