CRI DNA sequencing

CRI DNA sequencing

Back to Sequencing DNA This protocol will allow you to perform cycle sequencing usin ABI BigDye chemistry.

Materials

ABI Big Dye kit 3.1 part number 4336917

Miscellaneous Reagents

Protocol

Sequencing reaction setup

Using ABI Big Dye kit 3.1 stored at –20˚C aliquoted out, set up the following reaction to sequence plasmid DNA.

• DNA template = 1ul (200ng of plasmid DNA or Templiphi template DNA)
• Primer= eg.m13f/r(10ug/ml ,3.2pmole/ul) 1ul (make sure primer is correct for vector)
• Big Dye Mix3.1= 1ul
• 5xbuffer= 1.5ul
• Water= Xul
• Total= 10ul

Add all the components to a thin walled PCR tube (0.2ml thermo strip AB-0266, stores stocked, in 96well plate format) or 96 well plate and then spin in sigma 4K15 centrifuge @ 2000rpm for 10secs), cap tubes and LABEL THEM, spinning down should mix them. Perform cycle sequencing reactions on a PCR machine MJ Tetrad close and tighten lid use heated lid) and run the following program:

Protocol name: CRISEQ1

1. 96˚C for 10 sec
2. 50˚C for 5 sec (ramping at 1˚C/sec)
3. 60˚C for 4mins
4. goto step1 24 times
5. 4°C hold
6. End

Reactions need to be precipitated and run on automated sequencer.

Notes

1. List troubleshooting tips here.
2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
3. Anecdotal observations that might be of use to others can also be posted here.

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Contact

• mail to jamesdothadfieldatcancerdotorgdotuk
• This page was created by James Hadfield on 17 October 2006. I hope you found it useful.