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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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