Best Way to Sequence PCR Products?

Frank White fawhite at ix.netcom.com
Sat Aug 6 13:59:11 EST 1994


In <gustilo-0508942307040001 at ts7-14.upenn.edu> gustilo at pobox.upenn.edu writes: 

>What is the best way to sequence PCR products?
>1. Clean up the PCR product and sequence directly or,
>2. Ligate the PCR product into a vector and sequence the positive transforms?

Both ways work, but remember that you are potentially seeing two different things
with the two methods.  If you sequence the products directly, you see the "average"
of the population of PCR products produced, which is fine in most cases, unless you
expect to see several different things produced. Also, unless your PCR reaction gives
one strong band, or you gel purify the products, the sequence data will often not be
very clean.

If you clone first, you can usually get better quality sequence, but if you have 
several different species present, you'll only see what you cloned.  Also, remember
that PCR and especially the RT step (if used) can introduce errors, so if you sequence
cloned material, you should do several independent clones to make sure what you see is
representative of what's there, and you aren't looking at some single base changes
introduced by the amplification.  Hope this helps.

Frank White
fawhite at ix.netcom.com



>
>TIA for any advice
>
>--
>
>Standard Disclaimer:
>    I am not responsible for comments that I make after 3:00 A.M. or after 5 cups of double espresso, whichever comes first.
>
>gustilo at pobox.upenn.edu
>




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