I try to PCR up a 4kb fragment from a vector and with the simple ThermoFischer Taq I got the 4kb long fragment, however with the High Fidelity System the gele was empty... I need the sequence without any mistakes so somehow HF must work... Any tips how to optimalize? Thank you in advance!
Your target may be GC-rich....Add 1ul DMSO for 20ul reaction or using GC-rich specific buffer
Generally, high fidelity PCR implicates using of DNA polymerase with proof-reading activity. The latter, in turn, slows down the rate of DNA synthesis. Therefore, when high fidelity DNA polymerases are used, it is often necessary to increase elongation time, may be up to 1 min per kB. Otherwise, enzyme or enzyme mix works with insufiicient rate and couldn't elongate long DNA fragments.
I agree with Igor. Put at least 4 minutes of elongation time.
If it is stil not working, you may want to increase a little bit the Mg2+ concentration (but you will loose a little in fidelity)
SORRY, I CANNOT agree with all the guys above!!!!
Please check the extension speed of pfu!
From thermofisher,
https://tools.thermofisher.com/content/sfs/manuals/MAN0012888_Pfu_DNAPolymerase_ep0571_UG.pdf
"The optimal extension temperature for Pfu DNA Polymerase is 70-75°C. The recommended extension step is 2 min/kb at 72°C for PCR products up to 2 kb. For larger products, the extension time should be prolonged by 1 min/kb"
So, pfu should have a faster elongation rate (2kb/min) than taq which is 1kb/min
From NEB
https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase
"As a general rule, use extension times of one minute per 1000 base pairs (e.g. 3 minutes for a 3 kb product)"
Probably, I think the target sequence is GC-rich so that taq system was favorable but not pfu
Please use this website the check the GC-content of your target
http://www.endmemo.com/bio/gc.php
Thank you a lot! DMSO worked perfectly, i am just super slow responding here. Thank you All, especially Alex!
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