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Cloning a 3.5Kb fragment for sequencing


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#1 nimrod337

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Posted 03 August 2010 - 12:19 PM

edit: I just realized this would fit better in the cloning forum. If a mod could move it please, that would be awesome. Thanks.

Ok, I am running out of ideas. I am attempting to clone a fragment of a gene from potato and sequence it in attempts to design better primers to amplify the whole gene (promoter included). I can amplify the fragment pretty regularly using a platinum supermix PCR solution. I then isolate the fragment from a gel using a mo-bio kit. Then I A-tail the fragment, ligate into Pgem T-easy vector, transform via heat-shock, and plate on IPTG x-gal blue selective media. I got 5 white colonies (out of 100+ blue colonies). I performed PCR on the white colonies using T7 and SP6 primers (located on T-easy) This showed an insertion of appropriate length (3.5Kb) So then I grew the ecoli out, and isolated the plasmid, and sent it in to be sequenced using the T7 and SP6 primers. The sequencing returned back only vector sequence... And so to double check that the insert was truly in the plasmid, I ran a digestion using EcoRI. ran the results out on a gel, and observed only one band at 2.5Kb (supercoiled vector?) So, everything points to my insert being present, until I sequence or digest.
Is it possible that the colony PCR is amplifying the vector, and giving me a false positive? (if so, why is the colony still white when grown on IPTG x-gal plates?) Can someone offer any type of conclusion here? I am really stumped. Thanks

-A

p.s. what a great idea for a forum.

Edited by nimrod337, 03 August 2010 - 12:41 PM.


#2 ElHo

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Posted 04 August 2010 - 12:48 AM

Colony PCR is prone to false positives. I suggest you try a regular plasmid mini prep + restricition to screen your clones. Are you sure you have to additionally do A-tailing of your PCR-product? As far as I know the platinum PCR mixture contains Taq-polymerase which already adds a 3`A tail. But maybe Im wrong.

#3 nimrod337

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Posted 04 August 2010 - 06:22 AM

The Platinum supermix that I used to PCR the fragment has a proofreading enzyme in it (PFU I think.) And that is really a shame that the false positive is so consistently sized. I guess I will just have to attempt it once again... Cross your fingers.

-A




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