Colony PCR positive and Digestion negative????? - (Oct/10/2013 )
I tried to ligate an insert (1.5 kb) with an unphosphorilated vector (4.3kb) with 2 different sticky-end enzymes (KasI and XbaI).
Ligation yielded much more colonies in the positive (vector+insert) than in negative plate (only vector)
Colony PCR was positive for all 25 clones. Forward primer is in vector and reverse in insert.
Restriction digestion with the same enzymes (KasI and XbaI) showed only a single band with the expected size of the vector but no insert at all.
Has anyone any idea of what could be happening?
There was a paper published a number of years ago showing colony PCR's susceptibility for a high number of false-positives. The authors suggested that the false positives may be the result of the ligated vector + insert's inability to enter the bacterial cell. The correct ligation product would therefore be in your mixture of cells and may "stick" to the outside of your bacterial cell via a LPS. Who knows if this is the correct case, but I know I have had very little success with correctly identifying constructs via colony PCR. I usually avoid this procedure.
In your controls, do you mean that your vector only did not have ligase? It sounded like you just had ligation of your originally cut vector; however, it doesn't make any sense why your vector only would have fewer colonies...
your help is really appreciated.
That is an interesting hypothesis. However I have made many ColPCR and it is the first time that I face this problem.
About control. It is only vector with ligase. Since is a ligation with two different enzymes and unphosphorilated, colonies should only come from undigested vector, right?
Also the striking difference of colonies between positive and negative plate strongly suggest that the ligation worked!!!
Yes, your control sounds good. Your CIP treated vector with ligase should not religate unless your phosphatase is not working properly; especially with two non-compatible cohesive ends.
How close together are your two restriction sites? 3, 3, 12 bp?
Did you mean you have had success with colony PCR using these primers in the past?
You have a very interesting problem...
My restriction sites are separated 4.3kb in vector and 1.5Kb in insert. Is that what you are asking?
About the colony PCR primers, the primer in the vector I have used many times for other ColPCRs.The primer in the insert I was newly generated.
I never tried using this primers together, but the ColPCR seems to work because it gives an specific band of around 200bp
How far apart are your RE sites on your vector before digest?
Not all the times, colony PCR yields false positive results.
If you have used PCR amplified insert then I'd suggest you to use a reverse primer that you used earlier for amplifying insert as it would be quite long and there will be less chances of false positive reactions.
I had to face similar situation a couple of weeks ago. I used Forward primer from vector and lengthy reverse primer from the insert and problem was solved.
We have found it necessary to patch colonies arising on a transformation plate to fresh media, allow those to grow overnight, and sample them as template for colony PCR to avoid an undue number of false positives. Didn't know the observation of inordinately high false colony PCR positives was published anywhere, but in my lab, transferring colonies to fresh plates before using them in colony PCR is standard SOP, learned the hard way over many years of such experiments -- the extra day is well worth it...
sorry for my late answer but I was doing some tests.
Finally I solved the problem!!!!!
I just screened more colonies and found 2 colonies with my correct insert. I also confirmed by Sanger sequencing.
But the question remains in the air. All colonies are PCR positive and when I do Restriction enzyme digestion, most of them show the vector and a strange insert of around 500bp (my correct insert is 1500bp).
Where is this insert coming from and why gives more colonies than my correct insert?
Why the ColPCR is positive?
I really have no answer for that
Also many thanks to all of you