On 9 Feb 1997 21:56:44 -0000, derk at hubrecht.nl lost self-control and said:
>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 erro
>rrate of 1 per 270. To me this seems abnormal high. What are others
That is quite high. each cycle you will have an error rate of 1:10.000. After
25 cycles you will have +/- 1:400.
>How can I improve the fidelity of the amplification, except for
>using expensive high fidelity polymerases?
1. use lots of template (titrate the optimal concentration)
2. add more MgCl2 (idem)
3. add more nucleotides (but <200 microM)
4. run fewer cycles (titrate this as well)
5. depending on what you are doing: if you need the exact sequence:
better switch to a proofreading enzyme (twice as expensive)
>I used GoldStar DNA polymerase
>from Eurogentec and an Idaho Technologies air thermal cycler.
Thermocycler shouldn't be a problem. Taq polymerases vary highly in
quality from company to company.
>Thanks for your consideration.
met vriendelijke groeten,
>Derk ten Berge