Colony PCR - Next step problems (May/11/2009 )
Ok guys still me
So we were at the stage where I had a lot of colonies in the vector+ insert and no colonies in the vector alone. Since I did not gel purified the insert I should check by colony PCR for the insert.
I ran a first colony PCR straight from the plate and with primers only for the insert. I got tons of PCR product from all the 20 clones I have tried. I guess they are false positive from the plate I used during trasformation. The white was white and I used a colony from previous attempt for vector only and it was negative as well. However I guess they are all false positive since I do not think I have 100% colonies with the insert of interest, even if running a gel for the PCR product I can not see any band at lower size.
So I picked up then 16 clones from the same plate using a T7 and reverse from vector (I ran at 50C annealing and the band should appear at 1300, whist the band from insert primers previously was at 700). This time not a single one was positive. The problem is I do not have any positive control to check if the PCR has worked. What do you suggest to check for a positive control? Should I try on the ligase reacion to see if I have at least the product in the ligase mix I used to transfect the cells? Should I cary on with this PCR since is probably more "specific"? If not band at all from the clones straight from the trasformation plate is coming out should I think I have no the insert of interest with vector?
Any suggestion on this matter would be appreciated.
I do colony PCR simply picking with a tip a small portion of my colony and put it in my PCR mix with a final volume of 30 ul. During the PCR I prefrom a step at 95C for 10 mins and this should be enough to break the cells walls and get the template for the PCR. ANy suggestion?
Thanks
We have found colony PCR from a transformation plate to be riddled with false positives, so much so that we never do it any more -- we first pick the transformants to a fresh plate, grow them overnight, and do the PCR from there the next day.
You can use a vector-borne primer coupled with an insert-specific primer to screen your clones. This is what we always do, because a positive PCR done this way gives us two pieces of information: it confirms the presence of a correctly-sized insert, and it tells us the insert's orientation.
HomeBrew on May 12 2009, 12:48 PM said:
You can use a vector-borne primer coupled with an insert-specific primer to screen your clones. This is what we always do, because a positive PCR done this way gives us two pieces of information: it confirms the presence of a correctly-sized insert, and it tells us the insert's orientation.
We put the cells into 10-20 ul MilliQ, then heat that for 10 minutes in the cycler and take 1 ul from that into the PCR reaction. Much cleaner. We also us a plasmid-based primer and an insert-based primer to reduce false positives.
swanny on May 11 2009, 07:00 PM said:
HomeBrew on May 12 2009, 12:48 PM said:
You can use a vector-borne primer coupled with an insert-specific primer to screen your clones. This is what we always do, because a positive PCR done this way gives us two pieces of information: it confirms the presence of a correctly-sized insert, and it tells us the insert's orientation.
We put the cells into 10-20 ul MilliQ, then heat that for 10 minutes in the cycler and take 1 ul from that into the PCR reaction. Much cleaner. We also us a plasmid-based primer and an insert-based primer to reduce false positives.
But how to make a positive control?
We don't do a positive control at this point, because we know our vector-based primer works because we used it many times, and we know our insert-based primer works, or we wouldn't have an insert to clone. Also, any clone looking positive at this point is further confirmed by a restriction digest and (if required) sequencing of the insert. We always make our primers with a Tm of about 60C, so the pair should work.
If you get no positive colonies at this point, and you think it might be because the PCR failed rather than being a true result, you can do a plasmid extraction on a handful of clones, and check for an insert by restriction digest.
i remember perneseblue mentioning last time bout tis situation. it appears that some ligated DNA would stay on the plate and introduce the false positive.
Would suggest a subcloning prior to colony PCR.
After transformation, incubate the e. coli in 1 ml LB for 1-30 minutes. Pellet the cells and resuspended in fresh LB before plating. This can wash away free DNA and get rid of false positive problem.