unable to digest my recombinant plasmid! - (Jun/21/2007 )
hi to all,
i'm currently dealing with cloning in plant. after ligation and transformation of the ligation mixture into E.coli, i continue with the plating. There are colonies seen on the plate. Thus, i proceed with the confirmation test via PCR with specific primer. There are positive result as the recombinant plasmid carried the specific gene in it. However, when i try to digest the recombinant plasmid with the enzymes, the enzymes seem unable to cut.
please advice.
thank you
are you sure your restriction enzymes are ok? are the buffers you are using suitable for those enzymes, are you doing a double digestion? if yes, are those enzymes appropriate for double digestion. You might have a look to New England's Biolab's troubleshooting (http://www.neb.com/nebecomm/default.asp).
Good luck 
The problem is not the restriction enzyme.
Colony PCR initiated from transformation reactions can give rise to false positives if the primers anneal to the insert sequence due to excess DNA insert from the ligation reaction that is spread as part of the transformation reaction onto bacterial plates. Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL reaction (0.25 pmol/µL) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/µL). If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018 pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 e 10 molecules) of background insert are available for a 100 µL colony PCR assay.
To avoid this problem, at least one primer should anneal to the vector (Forward or Reverse primers are good for this). A negative control consisting of vector-only is also recommended for purposes of band identification.
thanks for your reply.i;m getting little confuse here and need your help to clarify it. are you trying to advise that i need to design another pair of primer for the vector and run a pcr reaction for the vector-only sample?
really appreciate for your help.thanks again
really appreciate for your help.thanks again
Your PCR got false positive signals.
Try another pair of primer which contains at least one strand targeted to your vector and do PCR again.
which enzyme did you used to digest your plasmid???
really appreciate for your help.thanks again
No. Tfitzwater meant the following
1 - if you used a primer pair that only amplified a region within the insert, it is very very possible that you will amplify leftover insert DNA sitting naked on the agarose plate. The signal that you get is not from plasmid DNA within the colony but from leftover DNA from the ligation. PCR is that sensitive.
2- to overcome this problem, you need to amplify the junction between the vector and insert. This bit of DNA is unique to the a successfully ligate plasmid. To amplfy this junction you will need a primer that binds to the insert and another primer that binds to the vector.
Try not to make the PCR product too big, Taq can only eficiency make PCR products below 1kb. Anything larger and you will have to optimise the PCR conditions which is a real hassle as you don't have a positive control (ie the plasmid you were looking for)
Hi people
I am struggling with the same problem for the past three months. I'm working on binaryvector which should be stuck with 800 bp insert. When i transform the ligation reaction i get colonies and do get positive result for colony pcr. I do include negative control but my result is still puzzling giving the correct size in negative control too. but in addition i will get bands of various other sizes. both in test and control things.
I was able to see good explanations for possibilities of this false positive results in this particular discussion. I'll be happy if any one of you could tell me how efficient my ligation reaction I can set to get the real positive reaction? I need your help terribly as I've got my project review within two weeks.
which enzyme did you used to digest your plasmid???
xba I and EcoRI
really appreciate for your help.thanks again
No. Tfitzwater meant the following
1 - if you used a primer pair that only amplified a region within the insert, it is very very possible that you will amplify leftover insert DNA sitting naked on the agarose plate. The signal that you get is not from plasmid DNA within the colony but from leftover DNA from the ligation. PCR is that sensitive.
2- to overcome this problem, you need to amplify the junction between the vector and insert. This bit of DNA is unique to the a successfully ligate plasmid. To amplfy this junction you will need a primer that binds to the insert and another primer that binds to the vector.
Try not to make the PCR product too big, Taq can only eficiency make PCR products below 1kb. Anything larger and you will have to optimise the PCR conditions which is a real hassle as you don't have a positive control (ie the plasmid you were looking for)
i tried to amplified the region between the vector and insert by designing a pair of specific primer that specificly flanked this region as i have the sequence for both the vector and insert. as a result, false positive samples were detected as from these false positive samples, only a few samples carried the PCR product (region between vector & insert). next, i planned to send these samples for sequencing for confirmation.