I am trying to amplify a large fragment of about 12k bp from genomic template. I have tried a few things and its not working using a long range kit. Any tricks that you recommend from people who did it?
Hi,
I use Go Taq Long from Promega successfully. Of course the PCR optimization procedure requires time. Offen the Crux was to find the optimal extension time.
I like these Phusion Polymerases very much because of their high polymerisation rates and the low mistake rate. I used the kit from NEB for some cloning.
As it was recommended by Maren you should try to adjust the optimum regarding amount of template, annealing temperature, cycle steps, with/without DMSO.
If you have still problems try different primers or amplify just the half of your template in one PCR and combine it with the second half afterwards.
Like Maren, we have had a lot of success with GoTaq Long from Promega.
I can just speak to NEB, and have no experience with other manufactures: In my hands, Q5 beats Phusion way and far. I had to do several reactions, especially long and GC rich ones that worked with Q5 but not Phusion, all else equal. Never the other way.
Just try 3-4 different polymerases you can find in your lab/building/institute. It depends on the PCR, I've had fantastic 10kb ampicons with Phusion when others didn't work, and I've had amplicons that didn't work with Phusion but worked beautifully with a home-made cheap crude polymerase....
Next to polymerases, try some other things like enhancers 5% DMSO (although it can increase mutation rate I found), or Betaine.. etc.
Also, do you know the sequence surrounding your target? Then try to find restriction enzymes that cut just outside your target, and pre-digest the gDNA before amplification. That will greatly reduce the secondary/tertiary etc structure of the DNA and can make a HUGE difference.
And, make sure your DNA isolation procedure is gentle and doesn't shear the DNA too much, that will affect long-range pcrs...
Consider a gentle method with proteinase K treatment.
Goodluck!
See also:
https://www.researchgate.net/post/Does_anybody_know_a_good_enzyme_for_high-fidelity_long-range_PCR
https://www.researchgate.net/post/Can_anyone_please_recommend_a_software_to_design_long_range_PCR_primers
https://www.researchgate.net/post/Long_Range_PCR_30_kb_Is_it_possible_from_human_DNA1
I forgot the name of enzyme but it was from Epicentre. I used that to amplify upto 12 kbp amplicon during my PhD. You have to give the amplification phase for 12 minute as 1minute for each 1kbp long amplicon. Other parameters should be same as smaller amplicon.
You can try with PrimeSTAR GXL DNA polymerase (TaKaRa).
https://www.nature.com/articles/srep05737
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