Subcloning: Loosing my favorite gene
(by hzhen from freeuk.com)
Fri Feb 16 08:04:33 EST 2007
On Thu, 15 Feb 2007 02:29:16 GMT, dk from no.email.thankstospam.net (DK)
>>in fact, if you gel-purify a larger band, and run it on a gel again, you may
>>be able to even see some of the lower size bands (if you have enough
>>material, of course!). There will not be a lot of it, but you don't need
>>much to successfully transform and obtain lots of clones.
>>It's just the way electrophoresis works... at the bottom of teh gel you will
>>only get the small stuff, but at the top you will get *mostly* large stuff,
>>but also a small % of "renegade" small stuff...
>Yes, stuff is retained "non-specifically" and is spread along the lane.
>But it is also noteworthy that at the bottom of the gel you will *also*
>find the large stuff.
I'm never quite sure what the large-sized band is. I think it is
possible to get dimer, but not too sure (see below).
To clarify to the original questioner - your normal plasmid prep
contains a mixture of supercoiled DNA, nicked DNA and denatured DNA
(if you used the alkaline lysis for DNA prep). They will run at
different speed. The supercoiled plasmid is normally the fastest
(although it also depends on EtBr concentration), the nicked (relaxed)
will run slower than a cut plasmid. The denatured one may be the one
with the big apparent size, although I'm not really sure about that.
Note that supercoiled DNA by itself can also run as different bands
depending on its superhelicity (I think). There is a good discussion
about plasmids running on gel in the Biochemistry book by Voet &
Voet,, haven't read it for some time, so my memory of it is a bit
hazy, but worth a read if you haven't read it before.
Anyway, if the original questioner has a protein that can be used as a
reporter (i.e. the GFP), it would make it easier to identify the clone
that has the GFP in it. That is, as I said before, if the GFP works
inside his favorite protein. If it does work, then personally I'd
just drop the phosphatase treatment step since you only need to see
one bright green colony amongst a thousand to know that you got a
possible right construct. If not done properly, phosphatase treatment
can be a cause of ligation failures, and thus the cause of much
unhappiness of many lab workers.
> Do a transformation with gel-purified "insert only"
>and you will find colonies belonging to the uncut plasmid from which
>the insert was excised. It is quite common, particularly when a well
>is nearly overloaded and small part of the sample spills out.
>I guess between being totally soluble and bound to agarose,
>DNA prefers latter.
More information about the Methods