McClean: Anneal and Extend - Revision history

Megan N McClean: /* Contact */

Megan N McClean — Wed, 19 Sep 2012 21:48:39 GMT

Contact

←Older revision		Revision as of 21:48, 19 September 2012
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	==Contact==		==Contact==
	<!--Change the information below to your info if you add a new protocol-->		<!--Change the information below to your info if you add a new protocol-->
-	*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 19 September 2012(EDT)'''	+	*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 19 September 2012 (EDT)'''

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

Megan N McClean: /* Contact */

Megan N McClean — Wed, 19 Sep 2012 21:48:29 GMT

Contact

←Older revision		Revision as of 21:48, 19 September 2012
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	==Contact==		==Contact==
	<!--Change the information below to your info if you add a new protocol-->		<!--Change the information below to your info if you add a new protocol-->
-	*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, ~~20 July 2011~~ (EDT)'''	+	*'''[[User:Megan N McClean\|Megan N McClean]] 14:01, 19 September 2012(EDT)'''

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

Megan N McClean: /* Protocol */

Megan N McClean — Wed, 19 Sep 2012 21:48:01 GMT

Protocol

←Older revision		Revision as of 21:48, 19 September 2012
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	==Protocol==		==Protocol==
-		+	# Design oligos so that they have ~20bp of overlap.
	# Dilute oligos to 25μM from the -80°C stock. Dilute into H<sub>2</sub>O.		# Dilute oligos to 25μM from the -80°C stock. Dilute into H<sub>2</sub>O.
	# For one reaction the PCR mix should be:		# For one reaction the PCR mix should be:

Megan N McClean: /* References */

Megan N McClean — Wed, 19 Sep 2012 21:47:26 GMT

References

←Older revision		Revision as of 21:47, 19 September 2012
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	==References==		==References==
-	Adapted from Maitreya Dunham's protocol (http://dunham.gs.washington.edu/sporulationdissection.htm) and the Botstein lab protocol (http://www.princeton.edu/genomics/botstein/protocols/Sporulation_and_Tetrad_Dissection.pdf)

	==Contact==		==Contact==

Megan N McClean: /* Notes */

Megan N McClean — Wed, 19 Sep 2012 21:47:16 GMT

Notes

←Older revision		Revision as of 21:47, 19 September 2012
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	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	*[[User:Megan N McClean\|Megan N McClean]]It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well.	+	*[[User:Megan N McClean\|Megan N McClean]] It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well.

	==References==		==References==

Megan N McClean: /* Notes */

Megan N McClean — Wed, 19 Sep 2012 21:46:53 GMT

Notes

←Older revision		Revision as of 21:46, 19 September 2012
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	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	~~#'''~~*[[User:Megan N McClean\|Megan N McClean]]~~''' When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit.~~ It ~~is absolutely infuriating, if not impossible,~~ to ~~try dissection on plates that are wet.~~	+	*[[User:Megan N McClean\|Megan N McClean]]It would probably be better to dilute oligos ordered for this purpose into water and not TE pH8 for the -80°C stock. However, I usually forget to do this and diluting from the TE stock 1:4 for 25μM and running the above protocol usually works really well.
-	~~#'''*[[User:Megan N McClean\|Megan N McClean]]''' Different tetrad dissection protocols call~~ for ~~using different enzymes to digest~~ the ~~ascus wall. Our protocol uses a β~~-~~glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail)~~. ~~Zymolyase~~, ~~another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at~~ the β-1~~,3-glucan linkages releasing laminaripentaose as~~ the ~~principal product. β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans)~~.	+

	==References==		==References==

Megan N McClean: /* Protocol */

Megan N McClean — Wed, 19 Sep 2012 21:45:15 GMT

Protocol

←Older revision		Revision as of 21:45, 19 September 2012
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	#Run in a thermocycler as follows (notice that we do not use many cycles):		#Run in a thermocycler as follows (notice that we do not use many cycles):
	#*94°C for 5 min		#*94°C for 5 min
-	#*Repeat the following three ~~(3)~~ lines 2-3 times	+	#*Repeat the following three lines 2-3 times:
	#**98°C for 10s		#**98°C for 10s
	#**53°C for 20s		#**53°C for 20s

Megan N McClean: /* Protocol */

Megan N McClean — Wed, 19 Sep 2012 21:44:57 GMT

Protocol

←Older revision		Revision as of 21:44, 19 September 2012
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	#**53°C for 20s		#**53°C for 20s
	#**72°C for 30s		#**72°C for 30s
-
	#*72°C 5 min		#*72°C 5 min

Megan N McClean: /* Protocol */

Megan N McClean — Wed, 19 Sep 2012 21:30:36 GMT

Protocol

←Older revision		Revision as of 21:30, 19 September 2012
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	#*33.5μL H<sub>2</sub>O		#*33.5μL H<sub>2</sub>O
	#Run in a thermocycler as follows (notice that we do not use many cycles):		#Run in a thermocycler as follows (notice that we do not use many cycles):
-	**94°C for 5 min	+	#*94°C for 5 min
-	**98°C for 10s	+	#*Repeat the following three (3) lines 2-3 times
-	**53°C for 20s	+	#**98°C for 10s
-	**72°C for 30s	+	#**53°C for 20s
-	**72°C 5 min	+	#**72°C for 30s
		+
		+	#*72°C 5 min

	==Notes==		==Notes==

Megan N McClean: /* Protocol */

Megan N McClean — Wed, 19 Sep 2012 21:29:39 GMT

Protocol

←Older revision		Revision as of 21:29, 19 September 2012
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	# Dilute oligos to 25μM from the -80°C stock. Dilute into H<sub>2</sub>O.		# Dilute oligos to 25μM from the -80°C stock. Dilute into H<sub>2</sub>O.
	# For one reaction the PCR mix should be:		# For one reaction the PCR mix should be:
-	**10μL 5X Takara Buffer	+	#*10μL 5X Takara Buffer
-	**4μL 2.5mM DNTPs	+	#*4μL 2.5mM DNTPs
-	**0.5μL Takara polymerase	+	#*0.5μL Takara polymerase
-	**1 μL 25μM forward oligo	+	#*1 μL 25μM forward oligo
-	**1 μL 25μM reverse oligo	+	#*1 μL 25μM reverse oligo
-	**33.5μL H<sub>2</sub>O	+	#*33.5μL H<sub>2</sub>O
	#Run in a thermocycler as follows (notice that we do not use many cycles):		#Run in a thermocycler as follows (notice that we do not use many cycles):
	**94°C for 5 min		**94°C for 5 min