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PCR using very long oligos !!!


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#1 lorenzo.costantino

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Posted 07 April 2015 - 04:19 PM

hi there,

 

I'm trying to PCR 1kb product using very long oligos. the For oligo is 118bp (18 anneal on the template and 100 are the tail) and a 69bp Rev (19 anneal on the template and 50 are the tail)

the template is a PCR fragment that I gel purified and is concentrated around 30ng/ul.

 

I'm trying to use the phusion taq from neb following the protocol but I got some of the PCR I need, but not all of them (the template is always the same, the only thing that changes in the different oligos is the tails)

 

any suggestions??

 

thanks!!



#2 bob1

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Posted 07 April 2015 - 06:40 PM

So - what have you tried?



#3 phage434

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Posted 08 April 2015 - 06:43 AM

Many problem can arise. The most serious is fold-back of the oligos on themselves, or annealing of two oligos. This can result in the pcr enzymes extending the 3' end of an oligo using the mis-annealed loop or dimer as a template, rather than your desired template. Once that happens, you no longer have any chance of extending that oligo by annealing it to your template. You need to make very certain that the 3' end of each of your oligos is as distinct as possible from annealing sites on itself or the other oligos in your pcr reaction. The homodimer and heterodimer tools at idtdna.com will help. Look especially for 3' end binding to a region with an available 5' extension on the complementary strand.



#4 lorenzo.costantino

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Posted 08 April 2015 - 01:26 PM

so I tried gradient PCR, and I got some of the pcr amplicon that I failed to get using the normal PCR but not all of them.

I tried touch down pcr with very similar results as the normal Phusion taq protocol.

I also tried TAIL-pcr and I got some of the missing amplicon, just are quite dirty with extra bands.

 

I cant change the sequence of this oligos since I'm targeting a specific genomic locus.

 

any help with a pcr protocol/taq that can solve the problem is highly appreciated!



#5 phage434

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Posted 08 April 2015 - 05:59 PM

You could try adding 5% betaine solution to your pcr reactions. It tends to equalize the Tm variations due to different GC content.






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