problems with restriction digestion

[Sudheendra Rao N R]

Hi,
Just to clarify!
How have to come to that conclusion that the bands u are seeing are extra?
sometimes insert might have restriction enzyme sites (i guess u are using
two enzymes on either side of MCS).
Try putting your insert virtually into the plasmid..using any plasmid
manipulation software (Vector NTI, DNAuser, plasmaDNA, Gentle etc)..cut it
with the enzymes you are about to digest it with (after circularizing
it)..and see how many bands u get (neb cutter is also okay)..
if you have done this job and still getting extra bands then
1. either your Enzymes are contaminated
2. of your mixture is receiving extra DNA from outside(check the digestion
components..especially water)
3. something else!!

sudheendra.

On Feb 8, 2008 11:39 AM, <chemophoto from gmail.com> wrote:

> 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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