IUBio

No Sequence

Stephen Stephen
Fri May 30 08:28:28 EST 1997


GMEACHAM at bmg.bhs.uab.edu wrote:
: I have sent a plasmid to be sequenced using a Perkin Elmer automated 
: system. They use the ABI PRISM dye terminator cycle sequencing kit.
:  I can PCR the sequence out (with low efficiency), but am unable to get 
: any sequence using the components above. The plasmid and ORF isn't GC 
: rich and my primers are desalted/deprotected. Any suggestions would be 
: helpful. Thanks in advance.

Most sequencing problems arise from poor quality template (in my
experience). How do you purify your plasmid DNA? I would recommend
Qiagen kits, followed by a good 10min spin to pellet resin, or a mini
CsCl preparation (see Saunders et al., 1987, in NAR, but don't worry
about the Ethidium bromide and isoprop extractions, go straight to the
pptn after the spin). The fact that you can only PCR the product out at
low efficiency could be due to either template quality or primer quality/
design. Check for palindromes in the primer sequence. I generally purify
my primers in the standard AMA/heat/dry (Beckman oligos) protocol, but
dialysis should be tried if you are worried about contamination with
salt etc. Also, a very basic error, but just in case, are your samples
resuspended in water? I have found TE is inhibitory in cycle sequencing
reactions.

Hope this helps


Steve





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