Sanger Sequencing - Big Dye Setup

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

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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