amackay at welchlink.welch.jhu.edu
Fri Jun 3 23:27:42 EST 1994
Jeffrey Wigle wrote about problems obtaining readable sequence from some,
but not other, Erase-a-Base clones. I had the same experience sequencing
a few kb of nested deletions made the same way. In my case, and I
suspect Jeffrey's as well, the problem is that the exo III sometimes
chews back past the "protecting" 3' overhang (eg BstXI). If it chews
back past the primer sequence (T7) but not to any essential sequence, you
have a viable construct of about the right size that you can't sequence.
Solutions: -- if there are enzymes in your MCS past BstX1, do a
diagnostic cut on candidates with one of them.
-- try lowering the exo III digestion temperature to 30 or even 22.
-- try a further-in primer, many vectors allow you to use an M13 + or -
primer instead of T7, T3, or SP6. This works surprisingly often.
-- Choose a 5' overhanging enzyme and fill with the thio-dNTPs that
Promega and others sell for the purpose. This works very well to
protect. For some reason, I found modified T7 or T4 pol (can't remember
which) worked much better than Klenow in protecting.
Good luck, Alastair
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