sequence data anomaly-PCR clone
jmorrey at sleepy.usu.edu
Thu Feb 13 13:17:50 EST 1997
We have sequenced cloned PCR products originating from synthetic
oligonucleotides, but are encountering an anomaly we have never seen
Question: Can this sequencing anomaly give any hints that may help us
solve our efforts to successfully clone a desired synthetic sequence?
Anomaly: Automated sequencing printout via cycle sequencing gave 30
bases of readable sequence with some homology to the expected sequence
(80%). The sequence was anticipated to be about 100 bases, but it
abruptly gave a straight line (no signal) after reading about 30 bases.
This has been repeatable between the same sequence and other sequences
using oligonucleotide templates and PCR primers synthesized at the same
time. Our DNA sequencing tech says that because we use the M13 primer,
we are sequencing across the multiple cloning site and there may be
"something" wrong with the MCS. Seems goofy, but she knows more about
these anomalies than I.
If you know that answer, then you not only help us with our problem,
but you answer a question that I don't think anyone would know.
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