In the past, I have obtained varying results with Phusion, but have been told there are other polymerases that can deal with the GC richness of some regions in genomic DNA. Do I really need to buy a special polymerase, or are there other ways to optimise for phusion PCR first. The largest fragment size I would be PCRing is 1.5 kb.
In the end, it would be great if I can show that my construct inserted exactly where we intended for it to insert - left and right homology arms were 1 kb each and I definitely want to have a forward primer in the artificial poly A tail (APAT) that we engineered right after our Gene of Interest (GOI)-T2A-Neo construct. This way we can show that our construct is present, the homology region of 1 kb is there and that right after our selected homology arm the sequence continues on as normal.
Any tips and advice would be greatly appreciated - Cheers.
Hi Cory,
from past experience, the addition of an ''enhancer'' such as betaine, ethylene glycol, 1,2-propanediol or Qiagen Q solution (you might have a qiagen kit with unused reactives in it) should do the trick with Phusion.
Otherwise, in recent months the OneTaq® DNA Polymerase from NEB has become my favorite because it works well, out of the box, with almost any templates, size and DNA extraction methods.
Good luck!
Thanks very much Nicolas, this answers my question perfectly. I'll try Phusion with the enhancers first, probably ethylene glycol as it is very cheap. Do you have any idea what the working concentration should be?
Thanks again,
Tony
Hi Cory,
I have linked a Biotechniques publication I have used to optimized my PCR during my Master. You'll have the recipe for betaine, ethylene and 1,2 propanediol. Q solution is a 5X buffer.
If you want something cheap, you can also try DMSO at 2,5 to 5% final. In combination with a gradient or a touchdown cycling it works for GC rich PCR but required more optimization in my hands.
Nicolas
I forgot to click add!
There you go
http://www.biotechniques.com/BiotechniquesJournal/2009/September/Enhanced-amplification-of-GC-rich-DNA-with-two-organic-reagents/biotechniques-173811.html
Hi Cory,
I am wondering if you could get an amplicon in the end? I am having the same problem and would be happy to get any advice.
Sofie
Hi Sofie,
I have been using phusion polymerase with the following protocol and have had some amazing results wit ethylene glycol.
For a 25 ul reaction, I use: 500 ug genomic DNA, 1.25 uL Primer F1 (10 uM stock) and 1.25 uL Primer R2 (10 uM stock), 5 uL of a 5X GC buffer (this buffer comes with phusion kit and has betaine in it), 0.5 uL dNTPs (10 mM each), 1.5 uL ethylene glycol and 0.25 uL polymerase all diluted to 25 ul with distilled water. The cycle is: 98 for 30 s, then 35 cycles of 98 for 5s, 58 for 30s and 72 for 30s - 1m (phusion can do 1kb/30s). A final extension of 72 for 5 min followed by 4C indefinitely. This basically resulted in me having a clean band when I had a smear of nothing. Give it a shot with ethylene glycol.
Good luck!
Hi Cory,
thank you for you quick answer, this sounds promising and I will directly test it with my samples as well.
Thanks again!
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