Cloning methylated DNA
blackhole at abuse.plus.com
Wed Nov 3 12:33:36 EST 2004
Historians believe that in newspost <Fe8id.179$Y2.4595 at news.tufts.edu>
on Wed, 3 Nov 2004, Mark <nospam at nspam.com> penned the following
>> hi everyone
>> my lab mostly use DH5alpha competent cells for transformation.
>> currently, i want to clone methylated dna. i read papers and most of the
>> people use XL1-blue competent cells. I am just wondering if it is possible
>> DH5alpha and i guess probably not. Am I right? Thanks!
Play safe. Do not use DH5alpha or XL1 Blue.
What you need is an mcrA, mcrBC, mrr minus E.coli plus the normal hsdR
minus i.e. A number that NEB offer for free or TOP10F' from Invitrogen
(my normal strain of choice) or some Stratagene cell lines
Have a look at an NEB catalogue in the appendix for the reasoning.
>Are you asking if DNA that is methylated and ligated to a vector can be
>used to transform DH5a? If so, I see no reason why not, (although there
>may be one). Most of the DNA that I prepare from various strains is
>assumed to be methylated, and I have routinely used DH5a as the
>recipient strain for my cloning procedures.
There is a difference between moving plasmids between E.coli strains and
cloning genomic DNA (that is say C methylated) from elsewhere into
>I guess you could be asking whether DH5a will maintain the methylation
>pattern. I think it probably will, as it is neither dam nor dcm minus
>(as far as I can recall).
It will not retain the methylation pattern of the original genomic
template being cloned. The cloned DNA will take on the methylation
characteristic of the E.coli i.e. dam dcm, hsd etc
DH5alpha is dam+ dcm+, as are virtually all standard laboratory E.coli
K12 strains. E.coli B is dcm minus as standard. If you want dam- or dam-
dcm- then look for strains beginning GM**** (free from NEB) or JM110
which is blue/white with an F'.
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
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