PCR THROUGH INVERTED REPEATS

Timo Hiltunen hiltunen at convex.csc.FI
Sat May 14 15:00:41 EST 1994


In <2qqdkl$lo6 at unicorn.ccc.nottingham.ac.uk> pdxrmb at vaxa.nott.ac.uk writes:

>This is a desperate appeal for help- since I have run out of ideas!!! My 
>problem is the amplification of a genomic insertion sequence containing
>four perfect inverted repeats. So far I have tried  Hot starts, Touchdown
>,DMSO, deaza-GTP and even SSB (single stranded binding protein) with no 
>success. Has anybody got any suggestions?? The problem seems to be how to
>destabilse the predicted secondary structure without impairing primer ann-
>ealing. Presumably if I can genearate a Deaza substituted copy of the seq-uencse PCR should be possible. All comments and suggestions are welcome, and thanks
>in advance. 

>R.Badge.

You probably have already done this, but:

design your primers to enable annealing temp:s of 72 C (or possibly higher)

and combine this with the other things you mentioned.

Timo Hiltunen



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