We are trying to amplify a 10 kb genomic fragment with Pfx (Invitrogen). The primers do work with the same enzyme when tested separately with other primers, generating smaller amplicons. The PCR for the large fragment seem to work, since a strong EtBr fluorescence can be seen in the gel, but it does not leave the well after electrophoresis. This problem was addressed here http://tools.invitrogen.com/content/sfs/brochures/711_021352_PlatPfxBf_bro.pdf, but adding SDS to the loading buffer is not working.
Hi Diogo,
I have performed PCR a bit smaller (5 a 8Kb), but I have not used Pfx.
However, for long PCR not only the enzyme is important. Also the anneling temperature of primer, the time of annealing and the length of primers, etc.
Moreover you can add at your reaction a mix of the two polymerases, Pfx and Taq to try improve the results.
Julio
Good Luck Pfx is notoriously slow and you will have great difficulty.
Try additives
http://www.staff.uni-mainz.de/lieb/additiva.html
DMSO 2-5% works well
More importantly try mixtures of polymerases
A DNA polymerase mix was made by combining 5 units of Taq DNA Polymerase and 0.2 units of Pfu DNA Polymerase (Cat.# M7741). The amplification reaction consisted of 1X Pfu DNA Polymerase Reaction Buffer, 250µM of each dNTP, 1µM each of sense and antisense oligonucleotide primers, 50ng of plasmid containing p300 cDNA and 1µl of the DNA polymerase mix in a total reaction volume of 50µl.
see
http://www.promega.ca/resources/articles/pubhub/enotes/long-pcr-amplification-of-p300-cdna-using-a-blend-of-taq-and-proofreading-pfu-dna-polymerase/?__utma=1.2145284040.1372213455.1372213455.1372213455.1&__utmb=1.1.10.1372213455&__utmc=1&__utmx=-&__utmz=1.1372213455.1.1.utmcsr=%28direct%29|utmccn=%28direct%29|utmcmd=%28none%29&__utmv=-&__utmk=49966630
Good luck
Try LongAmp Taq DNA Polymerase (blend of Taq and Deep VentR DNA Polymerases) www.neb.com
KOD-based polymerases (which Pfx is) have very low error rates but do not handle long amplicons very well. I would suggest using a Pfu-based polymerase (maybe Phusion) and increasing the dNTP concentrations.
More details:
http://www.biocompare.com/Application-Notes/42000-Comparing-Fidelity-And-Performance-Of-Proofreading-PCR-Enzymes/
The best system for long PCR is LongAmp® Taq DNA Polymerase (NEB)
(LongAmp® Taq DNA Polymerase is a unique blend of Taq and Deep VentR™ DNA Polymerases. The 3´→5´ exonuclease activity of Deep VentR DNA Polymerase increases the fidelity and robust amplification of Taq DNA Polymerase (1). LongAmp Taq DNA Polymerase offers two fold higher fidelity than Taq DNA Polymerase alone. A wide range of PCR products can be generated, up to 30 kb from lambda DNA or from human genomic DNA.)
Be sure to design your primers with a higher-than-usual Tm (e.g. 65-68°C) so you can reduce the amount of self annealing.
Have fun!
For a start amplifying large PCR fragments is not ideal as this will introduce undesired mutations in your sequence. I will suggest amplifying your 10kb fragment into 1.5kb overlapping fragments using a high fidelity enzyme like KOD Hot Start or Phusion DNA polymerase. I have protocols for these two polymerases that work perfectly well with my PCRs if you are interested.
I would avoid using any plain Taq for 10KB PCR specially if its for cloning. Try Pfu Turbo or PfU ultra (Agilent). 65C/min/kb elongation should work just fine.
I argee with Constance. KOD Hot Start seems especially useful for us to analyse difficult samples.
Hi, Diogo I have performed long pcr for a long period and I was able to amplify fragments ranging from 7000 to 20000 bp by using the Long PCR Enzyme mix (K0181). You can adjust the elongation time based on the amplicon lenght with a time increment each cicle. You can see the band on a 0.8% agarose gel run at 80V for 2-3 hour.
Hi Diogo, I have performed many long PCR, as you until 12Kb and the best system that I have worked is the Long Expand PCR System of Roche, it has three buffers systems to adjust the length of the fragment that you want amplify. You can see this band in a 0,4% agarose gel and run it at 50v. But is very important that you stain the gel after the run! Good luck.
Hi
I'm following the comments because I'm also trying to amplify a very long amplicon. I've got very interested in the protocol for overlapping PCR that Constance mentioned. Could you please, Constance, to share those protocols?
Thank you very much!
If you increase the enzyme concentration to 15 or 20 units per reaction and increase the elongation time according to specific enzyme activity but I recommend use a mix of taq platinum 2U + taq pfu 6U range 1:3 + pfu buffer + increased time elongation.
Hi Diogo, I performed long PCRs to amplifiy HIV proviral genome (about 9 kb) from genomic DNA using Expand Long Range (Roche). My protocol consisted in a outer PCR to amplify near full length HIV genome, followed by several nested PCRs generating overlapping fragments of about 3 kb. I found very useful to add 2% DMSO in outer PCR, then you could consider to extract genomic DNA using salting out method, avoiding commercial kits.
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