Hi,

My favourite DNA polymerase is the one from Clontech - SeqAmp DNA polymerase. I directly compared it with the NEBNext PCR MasterMix, KAPA HiFi and Phusion, and the one from Clontech showed the best performance. Though, I never used it for such long fragments, work mainly with the cDNA for RNA-Seq.

Best,

Michail

Hi Francois

I think Phusion DNA polymerase will work for 20kb gene and you can also try with Platinum SuperFi DNA Polymerase.

Dear Francois,

I do agree that KapaHifi for example as well as the phusion are great working enzymes. However, I would like to mention that from my experience there is no one and any polymerase out there. It rather depends on the template and the application. For instance long stretches amplified from genomic DNA will be in favor for one of the polymerases (Kapa Platinum or Phusion with adjusted DMSO concentration for instance), other quick amplifications using Q5 polymerase will also be great and super fast. In short, I would recommend to test a few enzymes to find the perfect one for your application. Good luck.

Hi,

The fragment is normally to long to survive typical genomic prep procedures. Polymerases mentioned by others may amplify something but the level of product will be minuscule not to mention errors.

Try something different. If you know what the sequence may be, try splitting it up in 3-4 pieces and then stitching it together. It may seem tedious but the other option would be to waste months of your time and $$$ of reagents before throwing in the towel.

Q5 is great but not above 5 kb. KOD HotStart Extreme can handle 10 kb easily. Phusion will most likely fail.

If you really want to be stubborn, try TOPO XL-2 PCR cloning kit. It is not only the yield but ability to fish out your DNA from a mixture. At this length, I would strongly recommend gel purification using proven systems. NEB can handle your gel purification needs but a complete kit for such length has to be optimized from A to Z. Think about it before dumping money on a single component.

Hi,

Fancois Kroll

I am also facing the same problem with my fragment of 11 Kb for genomic DNA. I have tried Phusion, but noting quite good came out. Currently Q5 is giving a little hope but it isn't consistent so far.

I think that strategy of Wieslaw Swietnicki to splitting your product to the little sequences with overlapping primers is also a nice suggestion to consider.

Still, if you want to go for the long range polymerase, TaKaRa claims the longest amplification so far (I guess!) of upto 30Kb genomic DNA fragment (I have no practical experience). Below is its link:

http://www.clontech.com/VN/Products/PCR/Long_PCR/LA_PCR_Kit

Try it!

Good Luck

Takara is not up to the par for subsequent cloning. Simply speaking, you get so many errors that fishing out your fragment AND putting it in a vector would be hopeless. If you have blue/whit screening (pUC19, SmaI cloning from a methylation-deficient strain), try it as blunt end cloning. You may get less than 1% of colonies which is not the best.

Your best bet for a combo is the TOPO XL-2 cloning kit. It can handle 20kb and the selection should work. The only disadvantage will be screening many colonies and fishing out error-free fragments.