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Colony PCR

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Colony PCR

Postby claudianisha » Fri Dec 02, 2011 1:59 am

Hey,

I'm having difficulties with my colony PCR.

I am working on a large gene (Pv ATPase 4). It's a plasmodium vivax gene. I amplified this ~4kb gene using Phusion polymerase. I was able to amplify the gene. I then purified it using the Qiagen Purification Kit and added 'A' overhangs to the product since Phusion produces blunt end products.

Once I added the 'A' overhangs (cycling conditions of 72 degrees for 25 minutes), I then cloned it into the TOPO vector (Invitrogen). After which I transformed 2.5ul of the cloned product into XL 10-Gold competent cells. I plated 50ul of it onto LB + Amp. There were many colonies the next day.

I picked out 6 random colonies and screened them using the M13 primers (forward and reverse) which is provided in the TOPO kit. I used an annealing temp of 60 degrees. There were no bands except for a band beneath the 250bp mark (I used 1kb Fermentas ladder; 250bp - 10 000bp). I then repeated the colony PCR with the 13 other colonies and this time I decreased the annealing temp to 50 degrees since the primer sequence of M13 is around this temperature. I also increased the extension time to 2mins 30 secs since my fragment is a very long one. Still, there was no band at my size. I had bands beneath the 250bp mark and 4 clones had a band at 1kb. My negative was clean

Could someone please advice me on what to do please

Thank you

Claudia
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Postby JackBean » Fri Dec 02, 2011 7:39 am

I don't know, what polymerase are you using for the colony PCR, but with M13 primers you will amplify your whole sequence (plus something extra) and since the usual speed is ~1000 bases per minute, I would try 4 minute long amplification.
Another possibility is to use other primers, maybe e.g. M13 forward + some reverse primer which binds in the beginning of your gene.
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Re: Colony PCR

Postby claudianisha » Fri Dec 02, 2011 7:59 am

I used Phusion polymerase for my colony PCR
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Postby JackBean » Fri Dec 02, 2011 11:09 am

based on the gel on this page http://www.neb.com/nebecomm/products/productm0530.asp it should be able to amplify 3.8 kbp even with 1 minute extension :(
I would probably try to isolate the plasmid and do some restriction to be sure, that it is there in the first place. Or use other primers.
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Postby canalon » Fri Dec 02, 2011 8:52 pm

This is a case where a positive control for the DNA of your colony PCR would be useful. Sometimes impurities are inhibiting the PCR and colony PCR is not always reliable. So you should check your PCR conditions (Phusion needs some modifications compared to other enzymes if I remember correctly) on some clean DNA (not colony), even without insert (and remember there are 2 different forward M13 primers kicking around) just to show that you should get something. If it works, you can then play with your colony PCR to see if your conditions needs to be tweaked (how many cells, does a separate boil of more cells followed by centrifugation helps compared to just putting cells in the PCR tube?).
Or just as Jack suggest, do a plasmid prep and check the size to see if you have a PCR problem or a cloning problem (The TELT protocol on 1ml cultures can give you DNA in less than a day if you start your cultures in the morning, as 1ml cultures will reach saturation in a matter of hours if started with enough of a colony).
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