Protocol/14

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Revision as of 01:44, 26 October 2010

TEAM ALBERTA

Protocol 14: 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^oC 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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@@ Line 11: / Line 11: @@
 {{Team:Alberta/beginMainContent}}
-Protocol 14: Fluorescent sequencing reaction
+Protocol 14: Fluorescent Sequencing Reaction
 Procedure:
-*In a 0.2 ml PCR tube add
+*In a 0.2ml PCR tube, add:
-** template 5.0 ul (200 ng/ul)
+{|
-** VF primer 1.0 ul
+|Template || 5.0ul (200 ng/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)
+|VF primer || 1.0ul
+|-
+|Dilute buffer || 2.5ul
+|-
+|BD sequence mix || 1.5ul
+|}
 * Mix well.
-* Select program 'seq-dye' on PTC 200 thermal cycler
+* Select program 'seq-dye' on PTC 200 thermal cycler.
-* Run program. It will take about 2 hrs.
+* Run program. It will take about 2 hours.
-* Remove tube from PCR machine and transfer 10 ul rxn mix to 1.5 ml Eppendorf tube. Add
+* Remove tube from PCR machine and transfer 10ul rxn mix to 1.5ml Eppendorf tube. Add:
-** 1.5 ul Blue NaOAc/EDTA
+{|
-** 40 ul 95% ethanol
+|Blue NaOAc/EDTA || 1.5ul
-* Let sit on ice 15 min
+|-
-* Centrifuge 10 min max speed
+|95% ethanol || 40ul
-* Should see a small blue dot at bottom of tube
+|}
-* Discard supernatant
+* Let sit on ice 15 minutes.
-* Wash pellet with 500 ul of 70% ethanol
+* Centrifuge 10 minutes at max speed.
-* discard supernatant and spin briefly to bring down the residual liquid. Draw liquid off with a P10 pipette tip. *Do not disturb the pellet.*
+** Should see a small blue dot at bottom of tube.
-* Air dry for 10 min
+* Discard supernatant.
-* 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.
+* 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. Rxns delivered before 2pm will normally be returned the next day.
 [[Team:Alberta/Notebook/protocols| Back]]
 {{Team:Alberta/endMainContent}}