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Amplification Problem


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5 replies to this topic

#1 febstein

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Posted 24 November 2003 - 02:39 AM

Hi, I have designed 20 nt-primers whose Tm are 59C and 60C, respectively and I am having deep trouble in getting the desired product whose expected size is supposed to be 1,6 kb. I have tried a Mg++ gradient from 1,5 to 2,0 M, but I only get unspecific products lower than the right size. Likewise, setting PCR at different annealing temperatures ranging from 55C to 60C was not effective. However, my PCR reagents and DNA template are working fine (checked that...). Could you please let me know whether you face the same problem and eventually guide me towards the answer ?

Many thanks.

#2 ale

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Posted 19 January 2004 - 08:59 AM

How did you design the primers?, using a DNA program?
What is your template, genomic DNA, plasmidic DNA?. It is obvious that your primers are not specific.
Have you checked homo and heterodimer formation?

#3 uaue

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Posted 20 January 2004 - 08:12 AM

why dont you try "hotstart" protocol and see if its more specific or you can try Taq Gold from Promega (its quite good this enzyme). Good luck but dont give up. thats what were all here for!!!!

#4 paenutella

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Posted 22 January 2004 - 08:52 AM

Why don't you try a touchdown PCR for increasing specificity? or even try to adjust Mg concentration(between 1 to 5 mM), expecially if you're working with genomic DNA and with a cheap polymerase?
Good luck..and DON'T GIVE UP! :D

#5 tfitzwater

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Posted 23 January 2004 - 03:05 PM

Actual PCR annealing temperatures are frequently placed 5C below the predicted Tm, so 55C may not be low enough.

#6 kant0008

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Posted 08 March 2004 - 09:40 PM

Hi,
you have quite a large segment to amplify, so what are your cycling times? Make sure you give your product enough time to be formed, you may have to go as much as 2mins for extension phase.




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