Sequencing artifacts

tkp2 at PO.CWRU.EDU tkp2 at PO.CWRU.EDU
Tue Sep 20 10:27:44 EST 1994


While sequencing Arabidopsis DNA (double stranded in Bluescript), I have
found that regions of the sequencing gel have bands across all four lanes.
I have been using Sequenase version 2.O (USB) and the use of dITP actually seems
to increase the problem. It seems that the problem may be resulting from
`pauses' by sequenase due to secondary structure.
To resolve such problems has anyone tried
a) a chase step using terminal deoxynucleotidyl transferase?
b) the use of thermostable polymerase kits?
Any suggestions would be appreciated.
Thanks,
Tilak Ponappa
Case Western Reserve University
tkp2 at po.cwru.edu




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