problems with restriction digestion - (Feb/07/2008 )
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
What buffer are you using? NEB recommends that a double digest can be done with buffer 3 + BSA. Are you adding BSA to the mix? I would do this digestion in higher volume, 50 ul for 1 ug of DNA. With a midi prep you could easily have 2+ ug of DNA in 1 ul. You should find out, or estimate from a gel. There is no reason to do these digestions one at a time. The extra dilution from doing the digestion in 50 ul may be helpful in handling contaminants from your alk prep. Are you sure you are simply not seeing the bands sometimes because of different amounts of DNA being present? Are the bands which are visible of different lengths?
http://www.neb.com/nebecomm/DoubleDigestCalculator.asp
could you show a gel pic to get a better of what is happening along with the respective sizes of the insert and vectors.
Do you have the complete sequence of vector and insert? ie, are you positive that BamHI and XbaI only cut once?
Yeah I do have the sequence and BamHI and XbaI cuts only once. I finally did a digest with EcoRI and BamHI and I got the right profile for my clone. So I thought I would send it for sequencing and see it the insert is there.
I still have no idea why XbaI/BamHI is not working.
Thanks for all the suggestions
http://www.neb.com/nebecomm/DoubleDigestCalculator.asp
As I was doing two digestions, I used Buffer B (roche) with BamHI and Buffer H with XbaI. No I don't use BSA.
I always see one extra band of the same size that should not be there (according to the sequence of the plasmid and insert).
Thanks a lot
XbaI can be dam methylated by overlap. Might this particular XbaI site be dam methylated?
Hi,
chemphoto i came to know that you used EcoRI and BamHI for double digestion,but i am using same enzymes of NEB company but they are not working.you used those enzymes in the past if yes please tell me the process how to do double digestion with these enzymes.
thanks in advance.
SVRREDDY,
Ph.D student,
Italy