To make an overexpression construct I amplified an 810 bp cDNA fragment and
subcloned it. Upon sequencing it turned out to have 3 nucleotide
substitutions, one c to t, one g to t and one t to a. This leads to an error
rate of 1 per 270. To me this seems abnormal high. What are others
experiences? How can I improve the fidelity of the amplification, except for
using expensive high fidelity polymerases? I used GoldStar DNA polymerase
from Eurogentec and an Idaho Technologies air thermal cycler.
Thanks for your consideration.
Derk ten Berge
Netherlands Institute for Developmental biology
Utrecht, The Netherlands
derk at hubrecht.nl