Sanger Sequencing - Big Dye Setup

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Sequencing reaction for Sequencing on ABI

In this reaction a PCR product (clean with ExoSap first) or part of a plasmid miniprep is used as template to incorporate marked base pairs for [Sanger sequencing]. After the reaction the sample can be run on an ABI sequencer to obtain the DNA sequence.

Do sequencing reaction:
1 μl Miniprep or clean PCR product
1 μl Primer (e.g. M13F) (10 μM)
5 μl H2O
2 μl BigDye sequencing mix (-20°C)
1 μl Sequencing buffer 5X (fridge, and yes, we use 5X to get a final concentration of 0.5X)

Run using “bigdye” program (in all machines under e.g. Gaby, Hilde or Simone)

Number of cycles Temperature Duration
1 X 96°C 1 min
40X 96°C 30 sec
50°C 15 sec
60°C 4 min
1X 72°C 4 min
7°C Inf

Possible alternative for insertion into genomic DNA (Not yet verified):
Horecka & Jigami 2000 Identifying tagged transposon insertion sites in yeast by direct genomic sequencing Yeast DOI: 10.1002/1097-0061(200007)16:10<967::AID-YEA597>3.0.CO;2-G

  • Isolate a lot of DNA
  • Do a BigDye reaction with 5ug genomic DNA in 20ul total volume
  • Use 90 cycles