Team:Alberta/Notebook/protocols/sequencing

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==Fluorescent Sequencing Reaction==


===Procedure:===

*In a 0.2ml PCR tube, add:
{|
|Template || 5.0ul (200 ng/ul)
|-
|VF primer || 1.0ul
|-
|Dilute buffer || 2.5ul 
|-
|BD sequence mix || 1.5ul
|}
* Mix well.
* Select program 'seq-dye' on PTC 200 thermal cycler.
* Run program. It will take about 2 hours.
* Remove tube from PCR machine and transfer 10ul rxn mix to 1.5ml Eppendorf tube. Add:
{| 
|Blue NaOAc/EDTA || 1.5ul
|-
|95% ethanol || 40ul
|}
* Let sit on ice 15 minutes.
* Centrifuge 10 minutes at max speed.
** Should see a small blue dot at bottom of tube.
* Discard supernatant.
* Wash pellet with 500ul 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 minutes.
* Place in -20<sup>o</sup>C freezer to be delivered to MBSU 4th floor microbiology M534. Reactions delivered before 2pm will normally be returned the next day.


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