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.