I understand that PFU taq have proofreading activity, therefore can reduce error during amplification, therefore it is recommended to use polymerase with pfu ability for fragments that will be used for cloning.
I need to amplify inserts of 1kb and 2kb for cloning purposes, either using Gibson, Slice or Aqua method. But our lab do not have pfu polymerase but we have long range taq polymerase which contain a mixture of normal taq and also pfu. I used it to amplify my fragments but unfortunately, the pcr products obtained are low in concentration no matter how i optimize it. Is the long range taq polymerase not suitable to amplify small fragments?
In my experience that should not be a problem. I always use KOD polymerase for cloning, no matter the size of the PCR product. Take another look at the primers (just in case), but sometimes it is just using another enzime for the PCR.
Hope it helps!
David
I agree with David that all enzymes should work well with short amplimers but sometimes the long read enzymes are more sensitive to Mg concentration than routine polymerases so if you have any impurity that removes Mg then it will have more effect on amplification so you could try diluting your dna more to dilute comtaminants and run more cycles to increase yield. Check the cycling parameters as some proof reading enzymes extend best at a lower temperature than Taq polymerases
In theory, it should work, but in practice it might take some trouble-shooting. Honestly, if I were you, I wouldn't waste my time trouble-shooting. Just buy a proof-reading taq like NEB Q5 https://www.neb.com/products/pcr-qpcr-and-amplification-technologies/q5-high-fidelity-dna-polymerases/q5-high-fidelity-dna-polymerases
1kb and 2kb fragments are not short. Low concentration of the products probably due to low primers efficiency. Cloning doesn' t requre huge amounts of DNA.
Since Pfu acts at the any nonextantion moment, lower the Pfu concetration, rise the primers and dNTP conc at the same time
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