Real Time PCR From Cell Culture - Revision history

Davebrid: added reference for calculating amounts from Ct

2011-03-29T00:42:21Z

added reference for calculating amounts from Ct

← Older revision		Revision as of 00:42, 29 March 2011
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	#Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL		#Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL
	#Start qPCR Machine using the machine in the [[qPCR - Lin Lab\|Lin Lab]] or the [[qPRC - Saltiel Lab\|Saltiel Lab]] machine		#Start qPCR Machine using the machine in the [[qPCR - Lin Lab\|Lin Lab]] or the [[qPRC - Saltiel Lab\|Saltiel Lab]] machine

			==Calculations==
			see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

	==References (Saltiel Lab)==		==References (Saltiel Lab)==

Davebrid: altered primer concentration and volumes for 384 well plate

2010-12-10T21:49:39Z

altered primer concentration and volumes for 384 well plate

← Older revision		Revision as of 21:49, 10 December 2010
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	*SyberGreen PCR Master Mix Applied Biosystems		*SyberGreen PCR Master Mix Applied Biosystems
	*96 well qPCR plate		*96 well qPCR plate
	*Primers (Dilute to 5 uM mixture of fwd and rev ~~and then make a working solution of 0~~.4 uL ~~+ 4.6~~ uL ~~water per reaction~~)		*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water)
	*Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html		*Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html

Line 38:		Line 38:
	===Plate Preparation===		===Plate Preparation===
	#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid		#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
	~~#Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.~~		#Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.
	#Get 96 well block and keep on rack. Do not touch bottom of plate.		#Add 5 uL template per well. If using a 384 well plate use 2.5uL
	#Add 5 uL template per well.		#Add 5 uL primer per well. If using a 384 well plate use 2.5uL
	#Add 5 uL primer per well.
	#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL		#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
	#Using a multichannel pipettor, add 10 uL Master mix to each well		#Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL
	#Start qPCR Machine using the machine in the [[qPCR - Lin Lab\|Lin Lab]] or the [[qPRC - Saltiel Lab\|Saltiel Lab]] machine		#Start qPCR Machine using the machine in the [[qPCR - Lin Lab\|Lin Lab]] or the [[qPRC - Saltiel Lab\|Saltiel Lab]] machine

Davebrid: added link to preparation of rna from tissue

2010-12-06T17:15:43Z

added link to preparation of rna from tissue

← Older revision		Revision as of 17:15, 6 December 2010
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	==Protocol==		==Protocol==
	===RNA Extraction===		===RNA Extraction===
	#Use RNEasy kit with Qiashredder. see [[Harvesting RNA from Cells grown in monolayer]]. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).		#Use RNEasy kit with Qiashredder. see [[Harvesting RNA from Cells grown in monolayer]] or [[Preparation_of_RNA_Samples_from_Mouse_Tissues]]. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
	#Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.		#Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
	#Store at -20 until RT reaction		#Store at -20 until RT reaction

Davebrid: added categories

2010-10-14T13:06:58Z

added categories

← Older revision		Revision as of 13:06, 14 October 2010
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	==References (Saltiel Lab)==		==References (Saltiel Lab)==
	~~<pubmed>~~18829989~~</pubmed>~~		PMID 18829989
	~~<pubmed>~~17008399~~</pubmed>~~		PMID 17008399
	~~<pubmed>~~17200717~~</pubmed>~~		PMID 17200717
	~~<pubmed>~~17192460~~</pubmed>~~		PMID 17192460
	~~<pubmed>~~16926380~~</pubmed>~~		PMID 16926380

			[[Category:Transcription]]
			[[Category:Expression]]
			[[Category:RNA]]
			[[Category:qPCR]]

Davebrid at 16:51, 20 August 2009

2009-08-20T16:51:58Z

← Older revision		Revision as of 16:51, 20 August 2009
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			__NOTOC__
	==Real Time qPCR==		==Real Time qPCR==
	===Materials===		===Materials===

Davebrid: /* Plate Preparation */

2009-07-21T18:49:53Z

Plate Preparation

← Older revision		Revision as of 18:49, 21 July 2009
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	===Plate Preparation===		===Plate Preparation===
	#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS		#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
	#Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.		#Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.
	#Get 96 well block and keep on rack. Do not touch bottom of plate.		#Get 96 well block and keep on rack. Do not touch bottom of plate.

Davebrid: /* RT-PCR Reaction */

2009-07-21T18:41:34Z

RT-PCR Reaction

← Older revision		Revision as of 18:41, 21 July 2009
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	===RT-PCR Reaction===		===RT-PCR Reaction===
	#Book PCR Machine for 2h		#Book PCR Machine for 2h
	#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water		#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water. All reagents are in the small red invitrogen box
	#Combine the following:		#Combine the following in a PCR tube:
	##8 uL RNA		##8 uL RNA
	##1 uL dNTP mix		##1 uL dNTP mix

Davebrid: /* RT-PCR Reaction */ added details about RT Reaction

2009-07-09T11:47:18Z

RT-PCR Reaction: added details about RT Reaction

← Older revision		Revision as of 11:47, 9 July 2009
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	===RT-PCR Reaction===		===RT-PCR Reaction===
	#~~Use~~ 8 uL of RNA ~~per~~ RT ~~reaction~~.		#Book PCR Machine for 2h
	#~~Use Superscript~~ RT~~-PCR kit from Invitrogen, following manufacturers instructions~~		#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water
	#Store cDNA at -20 until use		#Combine the following:
	~~#Typically dilute 5X in water (adjust for higher or lower expressing transcripts)~~		##8 uL RNA
			##1 uL dNTP mix
			##1 uL Random Hexamers
			#Put in PCR machine and run program '''RT 65C''' (takes 5 min)
			#In a separate tube add in the following order (for 10 reactions):
			##20 uL 10X RT buffer
			##40 uL 25 mM MgCl2
			##20 uL 0.1M DTT
			##10 uL RNAse Out
			#Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge
			#Sit on the bench for 2 min
			#Add 1 uL SuperScript II RT to each tube
			#Put in PCR machine and run program '''RT Reaction'''. After run (75 min) place on ice.
			#Add 1 uL RNase H and put in PCR machine and run program '''RT 37'''. Takes 20 min.
			#Store cDNA at -20 until use.

	===Plate Preparation===		===Plate Preparation===

Davebrid: /* Plate Preparation */

2009-05-28T12:09:26Z

Plate Preparation

← Older revision		Revision as of 12:09, 28 May 2009
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	===Plate Preparation===		===Plate Preparation===
	#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS		#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS
	#Prepare primer mix from 5 uM primer pair stocks ~~by adding~~ 0.4 uL primers + 4.6 uL water ~~per reaction~~		#Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.
	#Get 96 well block and keep on rack. Do not touch bottom of plate.		#Get 96 well block and keep on rack. Do not touch bottom of plate.
	#Add 5 uL template per well.		#Add 5 uL template per well.
	#Add 5 uL primer per well		#Add 5 uL primer per well.
	#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL		#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
	#Using a multichannel pipettor, add 10 uL Master mix to each well		#Using a multichannel pipettor, add 10 uL Master mix to each well

Davebrid: /* Plate Preparation */

2009-05-27T21:48:06Z

Plate Preparation

← Older revision		Revision as of 21:48, 27 May 2009
Line 29:		Line 29:
	#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL		#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
	#Using a multichannel pipettor, add 10 uL Master mix to each well		#Using a multichannel pipettor, add 10 uL Master mix to each well
			#Start qPCR Machine using the machine in the [[qPCR - Lin Lab\|Lin Lab]] or the [[qPRC - Saltiel Lab\|Saltiel Lab]] machine

	==References (Saltiel Lab)==		==References (Saltiel Lab)==