How can we directly sequence PCR-amplified DNA from agarose.

Shiao Y. Wang sywang at whale.st.usm.edu
Thu Apr 28 23:04:22 EST 1994


Wasun Chantratita (asmsi002 at CMU.CHIANGMAI.AC.TH) wrote:
: Dear Netters
: 	I am now trying to directly sequence PCR-amplified DNAs by cycle 
: sequencing. I was told that excess nucleotide and amplified primers can 
: interfere with the sequencing reactions. Thus,  PCR-amplified DNAs must 
: be purified by either a glass matrix or isopropanol precipitation. 
: However, would it be possible that we can cut a DNA band from perhaps low 
: MP agarose and subject to cycle sequencing directly. If yes, would you 
: please provide me that protocol or reference(s).

: 	Many thanks in advance
:         Wasun

Hello Wasun.
I gel-purify the PCR product (1.4 kb) using Prep-A-Gene kit from BioRad
and elute DNA in total of 20 ul TE. Using 2 ul of this as template, I got
acceptable sequence using the SequiTherm Cycle Sequencing Kit from
Epicentre Technologies. I used internal labeling w/ 32-PdATP. I'm going to
try doing PCR with one of the primers biotinylated and then separating
the template with magnetic beads coupled to streptavidin. I've heard that
it gives beautiful sequence info. Good luck.

Shiao Wang
University of Southern Mississippi




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