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Colony PCR


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#1 micky74

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Posted 11 May 2009 - 12:02 PM

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

#2 HomeBrew

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Posted 11 May 2009 - 06:48 PM

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.

#3 swanny

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Posted 11 May 2009 - 07:00 PM

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.

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.
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#4 micky74

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Posted 11 May 2009 - 10:39 PM

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.

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?

#5 HomeBrew

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Posted 12 May 2009 - 03:35 AM

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.

#6 hanming86

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Posted 15 May 2009 - 02:37 AM

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.
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#7 WHR

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Posted 16 May 2009 - 01:55 AM

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.




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