Protocol/14

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Protocol 14: Fluorescent Sequencing Reaction
 
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Procedure:
 
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*In a 0.2ml PCR tube, add:
 
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{|
 
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|Template || 5.0ul (200 ng/ul)
 
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|-
 
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|VF primer || 1.0ul
 
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|-
 
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|Dilute buffer || 2.5ul
 
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|-
 
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|BD sequence mix || 1.5ul
 
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|}
 
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* Mix well.
 
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* Select program 'seq-dye' on PTC 200 thermal cycler.
 
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* Run program. It will take about 2 hours.
 
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* Remove tube from PCR machine and transfer 10ul rxn mix to 1.5ml Eppendorf tube. Add:
 
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{|
 
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|Blue NaOAc/EDTA || 1.5ul
 
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|95% ethanol || 40ul
 
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|}
 
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* Let sit on ice 15 minutes.
 
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* Centrifuge 10 minutes at max speed.
 
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** Should see a small blue dot at bottom of tube.
 
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* Discard supernatant.
 
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* Wash pellet with 500ul of 70% ethanol.
 
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* Discard supernatant and spin briefly to bring down the residual liquid. Draw liquid off with a P10 pipette tip.
 
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** Do not disturb the pellet.
 
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* Air dry for 10 minutes.
 
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* 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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[[Team:Alberta/Notebook/protocols| Back]]
 
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Latest revision as of 17:13, 26 October 2010