Sanger Sequencing - Big Dye Setup
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Jump to navigationJump to searchSequencing 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) |
6.25 μl | H2O |
1 μl | BigDye sequencing mix (-20°C) |
0.75 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