Long Range PCR problem...

[SDK]

Hi Guys,

So I'm trying to amplify whole mtDNA of a few species using long-range PCR. I've found a conserved region and designed long (30mer) primers at 3 different sites which are about 120 bp and 500 bp apart. When I tested my primers by amplifying the small region in-between the primers I get beautiful bright bands for each pair. But when I use the reverse compliments of the primers to try for the whole 15 kbp, I get a smear and a few non-specific bands. I'm stumped. Primers are long, priming site is conserved, PCR is a 2 step with high Ta. Don't know what else to try. Ideas? PCR conditions as follows:

94C - 1 min
98C - 10 sec
68C - 15 min
(Last 2 steps X 33 cycles)
72C - 10

PCR recipe (25ul reaction):
dH2O - 12.25ul
10X buffer - 2.5ul
dNTP - 4.0
Primer F - 1.0
Primer R - 1.0
Taq - 0.25ul
Template - 4.0 ul (2-4ng)

Taq is Takara LA which is a high end long-range enzyme supposedly capable of amplifying up to 40 kbp. First picture is small amplicons proving priming sites are good, second image is the results for my long-range. HindIII ladder in second image.

[Youm Ug]

I have had the same issue in the past.
Few questions:
1) Who synthesized your primers?
2) Who synthesized your dNTPs?
3) What taxa is this?
4) Who made your ladder?
5) Who made the PCR cycler?

[SDK]

Hi Youm Ug,

Thanks for the response:

1) primers were made by IDT
2) the dNTP actually came with the taq so I assume it should be optimized for it.
3) the taxa is a platyhelminth (flat worm)
4) the HindIII ladder was made by NEB
5) the PCR Cycler is made by eppendorf.

Did you manage to resolve your issues?

[saimonara]

What type of reagents are you using? I have experience with long PCR with fragments that ranged from 5kb-12kb. When we first started trying to amplify these however, we were using commercially available long PCR kits and we were getting the smears that you mentioned.
We came up with our own cocktail of polymerases and conditions that gives you distinct band, and it's actually cheaper than the kits!
You can find the protocol and the publication about it here: http://simonuribe.com/Simon_Uribe-Co...lications.html

Reference: Uribe-Convers S, Duke JR, Moore JM, Tank DC. 2014. A long-PCR based method for chloroplast genome enrichment and phylogenomics in angiosperms. Applications in Plant Sciences 2: 1300063.

[Youm Ug]

Originally Posted by SDK

Hi Youm Ug,

Thanks for the response:

1) primers were made by IDT
2) the dNTP actually came with the taq so I assume it should be optimized for it.
3) the taxa is a platyhelminth (flat worm)
4) the HindIII ladder was made by NEB
5) the PCR Cycler is made by eppendorf.

Did you manage to resolve your issues?

Yes we managed to resolve it.
OK thats all very useful information.
One more thing before I tell you, how large are your loadings into the gel, and importantly what brand are your pipets?

[SDK]

Thank you Siamonara for your reply. Youm Ug, in the images you see I've loaded 5 ul PCR product. I don't recall what type of pipette tips we use, VWR perhaps.

[Youm Ug]

Originally Posted by SDK

Thank you Siamonara for your reply. Youm Ug, in the images you see I've loaded 5 ul PCR product. I don't recall what type of pipette tips we use, VWR perhaps.

Hmmmmm, a good scientist should always know the tools he works with intimately. That's probably why your experiments aren't working! Give your pipet a name. Treat your pipet like a woman...show some passion. Then I bet your reactions will start working.

Thanks,

Youm Ug

[saimonara]

Are you setting the reactions on ice? I find that setting everything on ice and then going to a preheated thermocycler reduces unspecific annealing. I always add a first step in my thermal profiles to make the thermocycler reach 95C and then pause until I hit start again. This way I can start the machine right before I add the taq and it's ready for me when I'm done.

[SDK]

Saimonara, thanks for the advice, I'll give the "hot start" a try. Youm Ug, I think you're just having fun at my expense. Don't need any of your ridiculous questions or crazy answers, go troll somewhere else please. However, I would love any more advice from others that have been in my shoes before, thanks.

[pmiguel]

How about another way?

Whole genome shotgun at a low X coverage?

Mitochondrial genomic DNA tends to be present in a high copy number compared to the nuclear genome. Someone did a MiSeq run in our facility on an amphibian. Mira says we got about 200x coverage on the mitochondrial genome.

Almost certainly would work with a HiSeq lane. If you could find some place that sold partial HiSeq lanes, you could get the job done for under $1000.

--
Phillip

[SDK]

Phillip,

Thanks very much for the reply. We have actually done that. We used the sequence generated to make denovo genomes which we used to develop primers for pop. gen. work. Now we want to 'kick it into high gear' as they say, sequencing many genomes (from different pops.) at once using PCR and Nextera. The only problem now is that the long-range PCR has been spotty, although priming sites are good. Since it is an AT-rich organism, the tech support team for the polymerase I'm using thought that perhaps secondary structures are forming, stopping the polymerase. I'm going to try to run a 2-step PCR at 72C to see if this may help...can't go much higher than that! Any other tips are welcome!

[pmiguel]

My experience with long range PCR did not go much past adding some DMSO to my PCR reaction. But it always seemed like up to 2 kb, you could do anything and it usually worked. Up to 4kb much more spotty, but with proofreading polymerase, it always worked. Above that? Exponentially more difficult.

So why not just pick a series of primers at closer intervals to cover the genome?

--
Phillip

[lkral]

I had done a number of long range PCRs up to 10KB from related species with a conserved pair of primers and I found that some enzymes and mixes worked and some did not. Funds permitting, you may want to try a sampling from different manufacturers. The one that worked best for me was the RANGER mix from Bioline but YMMV. However, for some species i still had to design additional primers and amplify in several shorter fragments.

[nusspli]

So inappropriate and chauvinistic.

Originally Posted by Youm Ug

Hmmmmm, a good scientist should always know the tools he works with intimately. That's probably why your experiments aren't working! Give your pipet a name. Treat your pipet like a woman...show some passion. Then I bet your reactions will start working.

Thanks,

Youm Ug