I try to amplify a 15kb fragment from a plasmid but can't manage to have any product yet. I tried to use Herculase II and condition suggested for product > 10kb but with no success. We don't have any kit to amplify such long DNA so I was wondering if some of you have a "home made" mix recipe of polymerases to help me to achieve this amplification as fast as possible?
Thanks for your help
Have you linearized the plasmid befor using it as a PCR template ? It makes a huge difference, since in its circular form it adopts supercoiling that prevents the Taq-polymerase to work.
Also, you should titrate the plasmid concentration down a lot. Don't even think about to use as high concentrations as from genomic DNA (i.e. 20ng or the like). If you have approx. 1000 copies of your plasmid in a 20µl PCR reaction, this should work. If you use much higher concentrations of the plasmid template, you got substrate inhibition and a lot of unspecific products instead of a single, specific band.
good luck, Michael
I think better to use a positive control along with around 10kb fragment and see if it works. High fidelty Polymerase should work for you theoretically. Good luck.
Hi, maybe you can ash for a free sample (like in the pdf). Good luck. Cheers, Nadine
http://www.neb-online.de/downloads/NEB_Polymerase_Fidelity_Article_2012.pdf
Have you linearized the plasmid befor using it as a PCR template ? It makes a huge difference, since in its circular form it adopts supercoiling that prevents the Taq-polymerase to work.
Also, you should titrate the plasmid concentration down a lot. Don't even think about to use as high concentrations as from genomic DNA (i.e. 20ng or the like). If you have approx. 1000 copies of your plasmid in a 20µl PCR reaction, this should work. If you use much higher concentrations of the plasmid template, you got substrate inhibition and a lot of unspecific products instead of a single, specific band.
good luck, Michael
First make sure that the primers designed to amplify 15 kp fragments are working, and the problem is solely due to the size of the product, you may order additional primers to amplify about 1 kb fragments corresponding to the ends of the 15 kb product. If you will be absolutely sure that your primers are fine, you may try different enzyme. I had good experience with Phusion High-Fidelity DNA Polymerase, sold by New England BioLabs, cat no. M0530S. to my view this is enzyme with he highest processivity. sure that you will be able to find this enzyme in Oxford, ( or even ask NEB sale rep for your area for a sample to try.
No product at all or no product at the expected size? Large plasmids are routinely amplified in variations of Quikchange site-directed mutagenesis, and 15kb is NOT so large that it should be exceptionally difficult. While linearizing your template DNA can help increase efficiency in standard PCR amplification, it usually doesn't make a difference in an "all-or-nothing" sense, in my experience. So worth a shot.
Have you checked for GC-rich stretches in your target sequence? You might need to add DMSO or betaine to overcome this. Also, try increasing the extension time to an interval between 10 and 15 minutes.
With such a long 15-kb PCR amplification, should the amount of PCR ingredient(s) (such as dNTPs) be adjusted accordingly?!
I did mutagenesis on 15.4 kb plasmid. I used reagents provided with Quick change II XL-Site Directed Mutagenesis Kit from Agilent Technologies, but the condition of the reaction described in Scott Shaun P et al paper (2002) [One-step Site Directed Mutagenesis of ATM....]. For point mutation primers were just desalted, for 3 points mutation introduced on 1 primer I have them HPLC purified. Of course, the outcome depends on Your template - repeats, secondary structures etc. Hope it helps!
As mentioned above Quick change II XL is good for doing this, I also used recently Herculase II, worked well. What Michael Rosemann mentioned might all help. I had sometimes bad experience with primer quality, also GC content is always an issue. If your primers have nearly the same Tm, what I normally do is to run 12 reactions on a gradient cycler without DMSO and 12 with DMSO, extension minimum 30s per kb. If this is not giving the correct band, then I change the primers (either same sequence purified or some base pairs extra to get a better GC content and more similar Tm). If you are in a rush it is faster to page purify the oligos yourself than to order HPLC quality from most companies.
You can please follow the links below:-
1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC373371/
2. http://2015.igem.org/wiki/images/9/9e/PCR_hifi.pdf
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