In an attempt to sequence gel-cleaned PCR fragments via cyclesequencing
we discovered, that apparantly a short exposure (ca. 5 min) to
short-waved UV light (355nm) was enough to make a sequencing impossible.
But anyway, all PCR fragments could be cloned in a T-overhang vector
(selfmade) and white transformands were obtained.
Now my questions:
1. If such a mutated fragment is cloned, will it be replicated normal
(high copy) and could sequencing of these clones lead to the right
DNA-sequence? Until now, no readable sequence could be obtained.
2. Could it be possible, that the cause of the problems is a radical
T-dimer mutation and crosslinking of the different fragments with each
Thanks for any input and suggestions.
email: banghans at inet.uni-c.dk