PCR cloning - how many clones must one sequence?
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Tue Mar 30 10:11:18 EST 2004
If your not doing so already, make sure to look at clones coming from
independent PCR reactions. If an error is introduced early on, of
course it will be represented multiple times when you isolate and
sequence clones derived from that reaction. It is very unlikely that
a random error would be introduced in two independent PCR reactions
though. When I am really interested in the sequence of PCR products
where the sequence is unknown, I usually do at least two independent
PCR reactions, and sequence at least two independent clones from each
if I am not sequencing the PCR product directly.
>We are cloning products from a nested PCR and single base variation
>occur in many clones. We use degenerated primers and taq polymerase
>for both PCR reaction. How many "alike" clones do we have to
>sequence in order to be sure that we have the correct sequence?
>Will it help if we use a proofreading enzyme instead of taq?
Michael L. Sullivan, Ph.D
Research Plant Molecular
U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706
(608) 264-5397 Phone
(608) 264-5147 Fax
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