Protocol/14

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Protocol 14: Fluorescent sequencing reaction
 
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Procedure:
 
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*In a 0.2 ml PCR tube add
 
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** template 5.0 ul (200 ng/ul)
 
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** VF primer 1.0 ul
 
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** dilute buffer 2.5 ul (said BD dilute buffer on tape of cap) in PCR/Sequencing box
 
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** BD sequence mix 1.5 ul (said BD)
 
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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 hrs.
 
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* Remove tube from PCR machine and transfer 10 ul rxn mix to 1.5 ml Eppendorf tube. Add
 
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** 1.5 ul Blue NaOAc/EDTA
 
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** 40 ul 95% ethanol
 
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* Let sit on ice 15 min
 
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* Centrifuge 10 min 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 500 ul 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. *Do not disturb the pellet.*
 
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* Air dry for 10 min
 
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* 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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[[Team:Alberta/Notebook/protocols| Back]]
 
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Latest revision as of 17:13, 26 October 2010