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Sequencing PCR Products
dIRECT Sequencing OF PCR Products
To obtain high quality sequencing data, it is
very important that the PCR reaction is specific and strong. If the PCR product is a smear on an agarose gel, or more
than one band is present, the likelihood of obtaining good sequence
data is low.
You must remove all PCR primers and unincorporated
nucleotides before the product is sequenced. Sequencing uses only one
primer instead of the two used in PCR. If you do not remove both primers,
you will get two sequences superimposed on each other that are not readable.
It is OK to use a PCR primer for sequencing as long as
it matches our conditions. Please see
Primer Guidelines
for more information.
Double check PCR concentration using an
analytical agarose gel. Over concentrating your template will not give you
a better sequence and could potentially interfere with neighboring researchers
samples on the instrument.
Applied Biosystems
has a useful manual for PCR Sequencing which can be downloaded as a PDF file. You
will need Acrobat Reader to complete the download.
Download PCR
Optimization -- Reaction Conditions and Components-pdf-file-
Another useful booklet is the Qiagen Guide
to Template Purification and DNA Sequencing.
Download the Qiagen Guide to Template Purification and DNA Sequencing
The recommendations for technical booklets
given above in no way represent an endorsement of either
ABI or Qiagen products.
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