DNA_Sequencing_Protocol

DNA_Sequencing_Protocol - 1/8 Sequence Reaction Protocol...

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1/8 Sequence Reaction Protocol from PCR or Plasmid Templates Updated 9/8/06 – ICG, 9/19/08 – JK 5x Sequence buffer 2.0 µl 20 µM primer 0.5 µl BigDye 3.1 1.0 µl **ALWAYS ADD LAST** PCR DNA 50-100 ng dH 2 O to 10 .0 µl Total Volume 10.0 µl 5x Sequence buffer 2.0 µl 20 µM primer 0.5 µl BigDye 3.1 1.0 µl **ALWAYS ADD LAST** plasmid DNA 100-500 ng dH 2 O to 10 .0 µl Total Volume 10.0 µl Note: The quantity of DNA does not seem to make a difference in the quality or the resolution of the read. This observation is based on several sequence reaction protocols using one plasmid with 2 primers. 100, 250, 350 or 500 ng DNA per reaction did not result in significant changes to the sequence length or quality. However, quantities of DNA may need to be modified for other plasmids as experience dictates. Run reactions using the following thermal cycle: 95 ° C for 5 min (1X) 95 ° C for 10 seconds, 50 ° C for 5 seconds, 60 ° C for 4 min (40X) hold at 4 ° C (or 10) until ready to purify. Samples can be stored for a few days at 4
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DNA_Sequencing_Protocol - 1/8 Sequence Reaction Protocol...

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