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Colony PCR Problem!!!!

Cloning

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5 replies to this topic

#1 andrea_UNSAM

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Posted 17 August 2011 - 11:22 AM

Hi, I nedd ask you a qestion: It's possible have pcr product en vector pRsetA with T7 promoter primer and specific primer for mi insert, even if my insert it's no here????? I had false positive (positive for this pcr, but no plasmid at all....., mean empty vector........:S) thanks!

Edited by andrea_UNSAM, 17 August 2011 - 11:48 AM.


#2 bob1

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Posted 17 August 2011 - 03:25 PM

Sure, if there is some contamination with the insert, of the plate that you are amplifying from. There could also be some contamination of your reagents if you are not very careful when handling plasmids, as they are typically very abundant and will persist in the environment for quite some time.

#3 andrea_UNSAM

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Posted 17 August 2011 - 04:19 PM

Sorry, the PCR is on plasmid intact (pRset isolated from another strain), I do that for negative control, and I sure it's no contaminated (sorry for my bad english). That is the reasson for mi surprise! Thank

#4 bob1

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Posted 18 August 2011 - 05:41 PM

Perhaps one of your PCR reagents, or even your negative control plasmid is contaminated?

It could also be that your negative control plasmid has a binding site for your specific primer - I suggest you get the full plasmid sequence and check this out.

#5 perneseblue

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Posted 25 August 2011 - 05:56 PM

also don't forget, if you don't use filter tip, it is possible to contaminate the barrel of your pipette.
May your PCR products be long, your protocols short and your boss on holiday

#6 labmonkey

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Posted 07 September 2011 - 08:17 AM

It's sounds a lot like contamination, but if you've done all the checks (H20 control etc) you might also want to check your primer annealing temperature. It your temp is too low you may get unspecific priming.

I use Primer 3 http://www.bioinform...primer3plus.cgi to design and check the predicted annealing temperature for my primers. I use 60oC as a default annealing temp and usually get good results.





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