low plasmid yields in dh5alpha
Jose de las Heras
(by josenet from tiscali.co.uk)
Thu Feb 21 18:54:24 EST 2008
"DK" <dk from no.email.thankstospam.net> wrote in message
news:Ewnvj.7456$8n4.3382 from newsfe07.lga...
> In article <6269mvF2288vvU1 from mid.individual.net>, "Jose de las Heras"
> <josenet from tiscali.co.uk> wrote:
>>"DK" <dk from no.email.thankstospam.net> wrote in message
>>news:Xj4vj.2179$fU6.161 from newsfe02.lga...
>>> In article <mailman.723.1203535541.2451.methods from net.bio.net>, Ed Siefker
>>> <ebs15242 from creighton.edu> wrote:
>>>>Hi. We've been going through our old plasmids and making
>>>>fresh stocks. We've found a few that we're not able to
>>>>miniprep and get reasonable concentrations (<50ng/ul).
>>> Are you sure these are not low/moderate copy number
>>> plasmids to begin with? WIth plasmids bearing pBR322
>>> origin, a "normal" miniprep almost never gives >50 ng/ul.
>>Yes, I'm sure... they went back to the usual >300ng/ul once the lysis
>>solution was taken care of.
>>They were all a series of constructs with the same backbone that we have
>>worked with regularly, so a lysis problem was the first thing we thought
>>off... and were right. If all troubleshooting worked like this! ;-)
> I don't understand how it is possible. If the lysis solution was to blame,
> it would affect the yield for all plasmids, not just some. The original
> post (quoted above) implied that the problem affected
> few of many.
I'm sorry, blame me for the misunderstanding.
In *my* case, the whole batch failed. That's why this is unlikely to be the
problem for the original poster.
I did mention it in my first reply as a "your problem reminded me of..."
kind of thing... as something basic that can easily be overlooked.
Where's my coat?
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