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assistance with PCR amplification please


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#1 jamesmhyu

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Posted 28 October 2010 - 01:57 PM

Hi Everyone,

I've been pushed to the edge of sanity with this PCR that I'm trying to do. I have to amplify the coding region (that means exons only, no introns and no UTRs) of a gene. In addition, I'm trying to add on two restriction cut sites to the ends as well. The gene itself isn't enormous (1.3 kb), but I've spent 4 weeks attempting to amplify and have ended up with very nice blank gels. One primer has a Tm of 72 (it is 27 bp long with a 59% GC content) while the other has a Tm of 65 (40 bp long, 33% GC). Since I only want to amplify exons only, I can't use alternative primers. I've never used primers longer than 40 bp and don't feel comfortable shortening the other below 27 bp. I've done qPCR to quantify expression levels of this gene in different cDNAs and the results are what is to be expected. I've selected the cDNAs that seem to have the highest expression levels for this gene.

My conditions are as follows: 95 degree melt, an annealing temp gradient between 55 - 65 (30 secs), and an extension temp of 72 (1:45 minutes). I'm using the Denville Taq and its respective Taq buffer (with Mg salts already mixed in).

Here's what I've tried in the last few weeks:
5% DMSO - I get a very very strong band at around 200 bp at all temperatures in my gradient. No other products. My positive control (beta-actin) shows up very strongly with no nonspecfic banding.
1 M betaine - No different from my normal PCR - very pretty ladders and nothing else. Positive control again shows up beautifully.
Pfx (high fidelity polymerase) - No different from my normal PCR. Weaker amplification of positive control.
Extension time of 2:30 - Random products show up, especially at my lower temperatures.

Any feedback or advice?

Sanity...slowly...disappearing...

James

Edit: I get weak primer dimers in all my PCR results, almost without fail.

Edited by jamesmhyu, 28 October 2010 - 02:00 PM.


#2 phage434

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Posted 28 October 2010 - 07:13 PM

What is the source of the DNA template? If it has low GC, then it might require a lower extension (not annealing) temperature. Try at 65C with 1.5x more time.

You could also look at the primer dimer and homodimer forming issues with your primers on the IDT web site, idtdna.com
What counts is whether there is high binding at the 3' end, with a 5' extension (either with a hairpin homodimer, or with a primer-dimer).

I would try lowering the annealing with a gradient down to 50C. Are those Tm's calculated only on the part of the primer binding initially, or on the entire primer, including the added RE sites? What counts is the binding initially, so the real Tm might be substantially below what you think.

#3 ElHo

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Posted 29 October 2010 - 12:59 AM

cDNAs are often fragmented, so maybe thatīs why you donīt get a full lenght amplicon. I once splitted the amplicon in two parts (pcr1 first half, pcr2 second half of the gene) and combined the pcr products in a third round of pcr using the outer two primers to get the full lenght transcript.

#4 jamesmhyu

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Posted 29 October 2010 - 07:12 PM

Thanks for the responses. I was finally able to amplify the product using fresh cDNA (rather than my old cDNA from 3 weeks ago). However, the band was still very weak and after purifying from the gel, the concentration was only 10 ng/uL. It's usable, but I'll be trying another PCR to get more product.

What is the source of the DNA template? If it has low GC, then it might require a lower extension (not annealing) temperature. Try at 65C with 1.5x more time.

The DNA template is cDNA made from an RNA extraction off of a tissue sample. The RNA concentration was initially pretty high (approx 2 ug/uL). I'll try a lower extension temperature in this go around and will let you know if it made a difference.

Are those Tm's calculated only on the part of the primer binding initially, or on the entire primer, including the added RE sites? What counts is the binding initially, so the real Tm might be substantially below what you think.

I had not thought of this. Thanks for this advice!

cDNAs are often fragmented, so maybe thatīs why you donīt get a full lenght amplicon. I once splitted the amplicon in two parts (pcr1 first half, pcr2 second half of the gene) and combined the pcr products in a third round of pcr using the outer two primers to get the full lenght transcript.


I was thinking about this today and talking about it with one of my colleagues. Would you have an overlap between the two chunks (almost like shotgun genomic sequencing) and would this overlap piece act as a "primer" when I stick my original primer mix back in there? Would any polymerase recognize that middle section as a primer? On the other hand, would you cut right down the middle, combine the PCR products into one tube, and use that as the template DNA. However, If I do this, I'm not sure how it will "glue" the two pieces together.

Thanks again for your input and I'll keep you all posted on how it goes.




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