problems with restriction digestion

chemophoto from gmail.com via methods%40net.bio.net (by chemophoto from gmail.com)
Fri Feb 8 01:09:32 EST 2008


Hi all,
Would really appreciate some help!!
I am trying to clone a gene and this is giving me a lot of problems.
When I started with an alkaline lysis prep of the plasmid and did a
restriction digestion with BamHI and XbaI I was getting extra bands
that should not be there. I than did a Midi prep of the plasmid and
did two sequential digestions with BamHI and XbaI and my problem
disappeared. I then went ahead with my cloning and finally got some
transformants. I did an alkaline lysis prep of my transformants for
screening  and did some restriction digestions and it seems I have a
few clones with the right profile.
So I went ahead and did a Midi prep (to get a pure prep) to
electroporate into my bug.  After I did my midi prep I did a digestion
with BamHI and XbaI (sequential) and again I am seeing extra bands
that should not be there (the same bands I saw before).

 I used a 20ul digest, 1ul restriction enzyme, 2ul buffer and 1ul of
plasmid DNA. I do not know the concentration of my plasmid DNA (didn't
nanodrop it yet). I used 1ul plasmid DNA based on the agarose gel
result of the midi prep.  I digested it for 2hrs at 37 and did an EtOH
pption and did the second digest.

I am wondering why I am getting extra bands. Am I using too much
enzyme? But I have used this amount of enzyme in digestions of 20ul
before and it worked.  Is there something else that I am doing
wrong??
I thought the reason why I got the extra bands before was as I was
using an alkaline lysis prep..which might have contaminants (RNA
etc).

Thanks a lot
Mary31


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