Hello everyone! I bought a KOD HotStart DNA Pol, from Merck Millipore. By reading the specifications and datasheet i could notice that this enzyme is capable of amplifying up to 21kb of plasmid DNA.
I'd like to amplify an entire plasmid of approximately 12kb, but besides the specification saying that the enzyme is able to do so, i cannot find any detailed protocol on how to do it. (There is a chart that came with the product that says that for PCR products longer than 3kb, i should set the extension time from 10s / kb to 25s / kb, which for my purpose, would mean a total extension time of 5 minutes per cycle, and i'm not too sure if it is going to work this way, since i've had bad experiences in the past by trying with long extension times. Although this was with other enzymes, i wouldn't like it happening again!)
So, if anyone has done anything similar before with KOD Hotstart and could share your PCR setup with me, i'd be really thankful!
:)
Greetings!
In last month I have experience with KOD One Master Mix 2X (Toyobo).
My target size for amplicons be apart in 5-20KB, and 3.5 min of elongation without addition time per cycle give good results for largest amplicons.
PCR condition which I used - 30 cycles of 94C 15 sec, 60C 5 sec, 68C 3,5 min (photo of electrophoresis below in png).
I was also curious to check the manual for fun on an amplicon measuring 1.5KB - 25 cycles of 5 sec with 98C, 5 sec with 55-68C and 3 sec 68C was enough.
However, I cannot be sure about the salt concentration in your case with KOD Hot Start polymerase, you can compare protocols, on Merck/Sigma-Aldrich's store my variation of chemistry under number KMM-101NV.
Hope my answer was helpful :)
P.S. sorry for electrophoresis quality :)
Sergey Baldaev Thank you very much for the answer!! Of course it was helpful! I'll compare between KOD One and HotStart to see if there are any major differences.
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