Why am I only getting smears for my genomic DNA long-distance PCR products? - It worked great with sheep BACs but failed miserably for genomic DNA (Oct/21/2008 )
This is my first posting so please be kind and I appreciate good explanations if you have experienced something like this or have any advices for me?
I have been using this kit called 'KOD Hot Start DNA polymerase kit' which provides excellent amplification of long-distance PCR products. It has worked well for my BACs (sheep bacterial aritificial chromosomes) and I have gotten excellent single dark bands between 6-12 kb everytime. However, I have not achieved anything much with my genomic sheep DNA even though I am using the same primer combinations.
I know that I would need more DNA with my genomic compared to my BACs and I have tried several concentrations but the results are quite inconsistent. Sometimes I get many un-specific bands but most often than not I get dark smears only. I have also increased my cycle (with BAcs I used 35, in gDNA I went up to 40..can I go higher than this???)
My BAC concentration was about 26ng/ 20ul PCR reactions, and genomic I've used a range of concentration: 50, 80, 100 and 150 ng/ 20ul PCR reactions
Thank you in advance for your feedback! I am happy to provide more information if requested
cheers,
meow
Hello,
did you amplify your fragments in bacs with the same primers as in your gDNA PCR?
I would consider making in order:
an annealing temperature gradient
a MgCl2 gradient from 1.5 to 4.5mM final concentration with optimal annealing temperature
and then temperature gradient with the optimum MgCl2 concentration
Did you try adding some BSA, DSMO ? else the quality of your gDNA might be at stake.
The complexity of genomic DNA is dramatically higher than that of your BAC. You probably don't want to hear this, but almost certainly the problem is the design of your oligos, which will need to be quite specific for amplifying large genomic DNA fragments. While some of the things suggested may rescue a failed PCR, the surest and quickest way is to redesign your oligos. Since they are now so cheap, design several for each end of the region and mix and match for success.
did you amplify your fragments in bacs with the same primers as in your gDNA PCR?
I would consider making in order:
an annealing temperature gradient
a MgCl2 gradient from 1.5 to 4.5mM final concentration with optimal annealing temperature
and then temperature gradient with the optimum MgCl2 concentration
Did you try adding some BSA, DSMO ? else the quality of your gDNA might be at stake.
Dear ph3no
Thanks for the prompt reply and sistematic order of optimization.
Yes, I'm running the same primers in my gDNA as those used for my BACs
I am currently doing a PCR with gradient annealing temp.. and Ill follow your order of optimization
I have also tried adding DMSO before. I'm pretty confident in my DNA. I've quantified them using the mass spec and the pico green method.
I've also used them for my shorter PCR products previously and they worked well (products of about 500bp- 3kb).In those reactions I used about 50ng/20ul reactions n I got good solid bands.
WIll update and follow up with you.. Thanks!
Dear phage 434
Thank you for the insight and I understand what you're getting at. I'm going to give the optimization steps a few more tries before actually going back to the primer drawing board. I'm working with a region of limited sequences (which is the main reason I'm sequencing the gene as the introns and 5 and 3'UTR are not published or sequenced yet) so there are not many regions I can design the primers from anyways. I have achieved quite successfully in the past using bovine and human aligned sequences to design my primers for sheep but it was rather tedious and time consuming.
I have tried sequencing some of my gDNA PCR products but they only work for small PCR length..between 500-800bp. So in a way, I am quite optimistic that with some optimization and tweaking they might just work..keep your fingers crossed for me
