PCR Amplification of DNA - Revision history

Reddj at 16:57, 8 June 2020

2020-06-08T16:57:11Z

← Older revision		Revision as of 16:57, 8 June 2020
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	==PCR Program==		==PCR Program==
	*For animals: use ~~program~~ denoted in [[Genotyping Program]]		*For animals: use programs denoted in [[Genotyping Program]]

	*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:

Reddj: updated

2020-06-08T16:56:49Z

updated

← Older revision		Revision as of 16:56, 8 June 2020
Line 9:		Line 9:
	==Protocol==		==Protocol==
	*Use the following volumes per reaction		*Use the following volumes per reaction
	::*~~Buffer,~~ 5 uL ~~of 10X buffer (Dave's fridge)~~		::*12.5 uL DreamTaq
	::*~~Primers, 10uL~~ of 1uM stock solution in water (both primers combined)		::*7.5 uL RNAse-Free water
	~~::*dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)~~		::*5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))
	~~::*Sterile water, 28 uL~~
	::*~~Template 1 uL~~		==PCR Program==
	:~~:*Polymerase 1 uL (turbo pfu found in "enzymes" box~~ in ~~freezer)~~		*For animals: use program denoted in [[Genotyping Program]]

	*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:

Reddj: updated

2020-06-08T16:49:40Z

updated

← Older revision		Revision as of 16:49, 8 June 2020
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			==SOP==
			*[[SOP - Electrophoresis]]

	==Materials==		==Materials==
	*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)		*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 0.4uM-1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
	*dNTPs ~~– dilute to 2 mM each in water~~, ~~make single use aliquots (50 uL aliquots~~..~~. 10uL of each dNTP, 460uL water)~~		*DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye https://www.thermofisher.com/order/catalog/product/K1081?ICID=search-product#/K1081?ICID=search-product
	*~~Template – generally 1uL or less of a plasmid miniprep~~		*RNAse-Free water - comes in DreamTaq kit
	*Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

	==Protocol==		==Protocol==

Darlanda at 16:02, 5 May 2009

2009-05-05T16:02:40Z

← Older revision		Revision as of 16:02, 5 May 2009
Line 1:		Line 1:
	==Materials==		==Materials==
	*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined		*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
	*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL)		*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
	*Template – generally 1uL or less of a plasmid miniprep		*Template – generally 1uL or less of a plasmid miniprep
	*Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.		*Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.
Line 7:		Line 7:
	==Protocol==		==Protocol==
	*Use the following volumes per reaction		*Use the following volumes per reaction
	::*Buffer, 5 uL of 10X buffer		::*Buffer, 5 uL of 10X buffer (Dave's fridge)
	::*Primers, 10uL of 1uM stock solution in water (both primers combined)		::*Primers, 10uL of 1uM stock solution in water (both primers combined)
	::*dNTPs, 5uL of 2 mM		::*dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)
	::*Sterile water, 28 uL		::*Sterile water, 28 uL
	::*Template 1 uL		::*Template 1 uL
	::*Polymerase 1 uL		::*Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer)

	*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''DAVETD''') as follows:
	:#1 min at 94		:#1 min at 94
	:#30s at 65		:#30s at 65

Davebrid: /* Protocol */

2009-05-05T13:25:04Z

Protocol

← Older revision		Revision as of 13:25, 5 May 2009
Line 6:		Line 6:

	==Protocol==		==Protocol==
	#Use the following volumes per reaction		*Use the following volumes per reaction
	::*Buffer, 5 uL of 10X buffer		::*Buffer, 5 uL of 10X buffer
	::*Primers, 10uL of 1uM stock solution in water (both primers combined)		::*Primers, 10uL of 1uM stock solution in water (both primers combined)

Davebrid: /* Protocol */

2009-05-05T13:24:30Z

Protocol

← Older revision		Revision as of 13:24, 5 May 2009
Line 15:		Line 15:

	*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:
	#1 min at 94		:#1 min at 94
	#30s at 65		:#30s at 65
	#2 min/kb at 72		:#2 min/kb at 72
	#30s at 94		:#30s at 94
	#30s at 63 then -0.5/cycle		:#30s at 63 then -0.5/cycle
	#2 min/kb at 72		:#2 min/kb at 72
	#Repeat steps 4-6 28 times		:#Repeat steps 4-6 28 times
	#30s at 94		:#30s at 94
	#30s at 45		:#30s at 45
	#11 min at 72		:#11 min at 72
	#~~hold~~ at 4 until ready		:#Hold at 4 until ready
	*Purify PCR product if necessary using Qiagen kit (Add 5x PB)		*Purify PCR product if necessary using Qiagen kit (Add 5x PB)

Davebrid at 13:09, 5 May 2009

2009-05-05T13:09:48Z

← Older revision		Revision as of 13:09, 5 May 2009
Line 15:		Line 15:

	*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:
	##1 min at 94		#1 min at 94
	##30s at 65		#30s at 65
	##2 min/kb at 72		#2 min/kb at 72
	##30s at 94		#30s at 94
	##30s at 63 then -0.5/cycle		#30s at 63 then -0.5/cycle
	##2 min/kb at 72		#2 min/kb at 72
	##Repeat steps 4-6 28 times		#Repeat steps 4-6 28 times
	##30s at 94		#30s at 94
	##30s at 45		#30s at 45
	##11 min at 72		#11 min at 72
	##hold at 4 until ready		#hold at 4 until ready
	*Purify PCR product if necessary using Qiagen kit (Add 5x PB)		*Purify PCR product if necessary using Qiagen kit (Add 5x PB)

Davebrid at 13:09, 5 May 2009

2009-05-05T13:09:15Z

← Older revision		Revision as of 13:09, 5 May 2009
Line 12:		Line 12:
	::*Sterile water, 28 uL		::*Sterile water, 28 uL
	::*Template 1 uL		::*Template 1 uL
	::Polymerase 1 uL		::*Polymerase 1 uL

	*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:

Davebrid at 13:08, 5 May 2009

2009-05-05T13:08:59Z

← Older revision		Revision as of 13:08, 5 May 2009
Line 7:		Line 7:
	==Protocol==		==Protocol==
	#Use the following volumes per reaction		#Use the following volumes per reaction
	*Buffer, 5 uL of 10X buffer		::*Buffer, 5 uL of 10X buffer
	*Primers, 10uL of 1uM stock solution in water (both primers combined)		::*Primers, 10uL of 1uM stock solution in water (both primers combined)
	*dNTPs, 5uL of 2 mM		::*dNTPs, 5uL of 2 mM
	*Sterile water, 28 uL		::*Sterile water, 28 uL
	*Template 1 uL		::*Template 1 uL
	*Polymerase 1 uL		::Polymerase 1 uL

	#Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:		*Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:
	##1 min at 94		##1 min at 94
	##30s at 65		##30s at 65
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	##11 min at 72		##11 min at 72
	##hold at 4 until ready		##hold at 4 until ready
	#Purify PCR product if necessary using Qiagen kit (Add 5x PB)		*Purify PCR product if necessary using Qiagen kit (Add 5x PB)

Davebrid: Created page with '==Materials== Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined dNTPs – dil...'

2009-05-05T12:53:04Z

Created page with '==Materials== *Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined *dNTPs – dil...'

New page

==Materials==
*Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined
*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL)
*Template – generally 1uL or less of a plasmid miniprep
*Polymerase – use Pfu Turbo for cloning and Taq for noncloning. Use appropriate buffer.

==Protocol==
#Use the following volumes per reaction
*Buffer, 5 uL of 10X buffer
*Primers, 10uL of 1uM stock solution in water (both primers combined)
*dNTPs, 5uL of 2 mM
*Sterile water, 28 uL
*Template 1 uL
*Polymerase 1 uL

#Run PCR Program. Normally use touchdown PCR ('''DAVETD''') as follows:
##1 min at 94
##30s at 65
##2 min/kb at 72
##30s at 94
##30s at 63 then -0.5/cycle
##2 min/kb at 72
##Repeat steps 4-6 28 times
##30s at 94
##30s at 45
##11 min at 72
##hold at 4 until ready
#Purify PCR product if necessary using Qiagen kit (Add 5x PB)

PCR Amplification of DNA - Revision history

Reddj at 16:57, 8 June 2020

Reddj: updated

Reddj: updated

Darlanda at 16:02, 5 May 2009

Davebrid: /* Protocol */

Davebrid: /* Protocol */

Davebrid at 13:09, 5 May 2009

Davebrid at 13:09, 5 May 2009

Davebrid at 13:08, 5 May 2009

Davebrid: Created page with '==Materials== *Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined *dNTPs – dil...'

Davebrid: Created page with '==Materials== Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined dNTPs – dil...'