Protocol/14

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Revision as of 00:43, 26 October 2010

TEAM ALBERTA

Protocol 14: Fluorescent sequencing reaction

Procedure:

In a 0.2 ml PCR tube add
- template 5.0 ul (200 ng/ul)
- VF primer 1.0 ul
- dilute buffer 2.5 ul (said BD dilute buffer on tape of cap) in PCR/Sequencing box
- BD sequence mix 1.5 ul (said BD)
Mix well.
Select program 'seq-dye' on PTC 200 thermal cycler
Run program. It will take about 2 hrs.
Remove tube from PCR machine and transfer 10 ul rxn mix to 1.5 ml Eppendorf tube. Add
- 1.5 ul Blue NaOAc/EDTA
- 40 ul 95% ethanol
Let sit on ice 15 min
Centrifuge 10 min max speed
Should see a small blue dot at bottom of tube
Discard supernatant
Wash pellet with 500 ul of 70% ethanol
discard supernatant and spin briefly to bring down the residual liquid. Draw liquid off with a P10 pipette tip. *Do not disturb the pellet.*
Air dry for 10 min
Place in -20 C freezer to be delivered to MBSU 4th floor microbiology M534. Rxns delivered before 2pm will normally be returned the next day.

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