Jacobs:Protocol Real-Time PCR - Revision history

David Hoey: /* Overview */

2009-11-30T21:36:03Z

Overview

←Older revision		Revision as of 21:36, 30 November 2009
Line 1:		Line 1:
	==Overview==		==Overview==

-	Protocol for ~~Real-Time~~ PCR	+	Protocol for RT PCR

	==Materials==		==Materials==

Ashley Chou at 02:14, 17 June 2008

2008-06-17T02:14:54Z

←Older revision		Revision as of 02:14, 17 June 2008
Line 25:		Line 25:

	Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):		Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):
-

	For one reaction		For one reaction

Christopher Jacobs: /* Procedure */

2008-05-07T22:39:12Z

Procedure

←Older revision		Revision as of 22:39, 7 May 2008
Line 45:		Line 45:
	#Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.		#Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.
	#Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):		#Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):
-	##20ºC 10 min	+	#*20ºC 10 min
-	##37ºC 30 min	+	#*37ºC 30 min
-	##99ºC 5 min	+	#*99ºC 5 min
-	##4ºC 5 min (but set to 1 hr)	+	#*4ºC 5 min (but set to 1 hr)
	#Freeze at -20ºC until further use.		#Freeze at -20ºC until further use.

	* Real Time PCR		* Real Time PCR
-	# Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in triplicate)~~. Total Volume 4.75 μl~~:	+	# Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in triplicate):
-	## Taqman PCR Master Mix 2.5 μl	+	#* Taqman PCR Master Mix 2.5 μl
-	## 20X Primer & Probe 0.25 μl x (N+1) Reactions	+	#* 20X Primer & Probe 0.25 μl x (N+1) Reactions
-	## RNase free H2O 2 μl	+	#* RNase free H2O 2 μl
		+	#*Total Volume 4.75 μl
	#Add 4.75 μl of mixed reagents to each well corresponding to specific genes (in this lab, we are only looking at 18S; the wells are A1-A12 and B1-B15) in the 384-well plate.		#Add 4.75 μl of mixed reagents to each well corresponding to specific genes (in this lab, we are only looking at 18S; the wells are A1-A12 and B1-B15) in the 384-well plate.
	#Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.		#Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.

Christopher Jacobs: /* Procedure */

2008-05-07T21:51:48Z

Procedure

←Older revision		Revision as of 21:51, 7 May 2008
Line 42:		Line 42:
	==Procedure==		==Procedure==
	* Reverse Transcription		* Reverse Transcription
-	#In PCR reaction tube, add 20 μL of Master Mix (~~from Step 1~~) and 20 μL of RNA sample (appropriate amount containing 3 μg of RNA and then q.s. to 20 μL). Centrifuge briefly (~10 sec).	+	#In PCR reaction tube, add 20 μL of Master Mix (above) and 20 μL of RNA sample (appropriate amount containing 3 μg of RNA and then q.s. to 20 μL). Centrifuge briefly (~10 sec).
	#Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.		#Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.
	#Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):		#Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):
-	* 20ºC 10 min	+	##20ºC 10 min
-	*37ºC 30 min	+	##37ºC 30 min
-	*99ºC 5 min	+	##99ºC 5 min
-	*4ºC 5 min (but set to 1 hr)	+	##4ºC 5 min (but set to 1 hr)
-		+	#Freeze at -20ºC until further use.
-	##Freeze in -20ºC until further use.	+

	* Real Time PCR		* Real Time PCR
-	# Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in ~~triplicates~~):	+	# Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in triplicate). Total Volume 4.75 μl:
	## Taqman PCR Master Mix 2.5 μl		## Taqman PCR Master Mix 2.5 μl
	## 20X Primer & Probe 0.25 μl x (N+1) Reactions		## 20X Primer & Probe 0.25 μl x (N+1) Reactions
	## RNase free H2O 2 μl		## RNase free H2O 2 μl
-	~~Total Volume 4.75 μl~~
-
	#Add 4.75 μl of mixed reagents to each well corresponding to specific genes (in this lab, we are only looking at 18S; the wells are A1-A12 and B1-B15) in the 384-well plate.		#Add 4.75 μl of mixed reagents to each well corresponding to specific genes (in this lab, we are only looking at 18S; the wells are A1-A12 and B1-B15) in the 384-well plate.
	#Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.		#Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.
	#Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.		#Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.
-	#For 18S (house keeping gene) ~~Standards~~, prepare five 1.5 ml microcentrifuge tubes and label them 1, 2, 3, 4, 5. Add 9 μl RNase free ~~H2O~~ in all five tubes. Add 1 μl of cDNA (use any one of Sample cDNA) in #1. Mix well and transfer 1 μl from tube 1 to tube 2 (10-fold dilution). Mix well and transfer 1 μl from tube 2 to tube 3. Mix well and transfer 1 μl from tube 3 to tube 4. Mix well and transfer 1 μl from tube 4 to tube 5.	+	#For 18S (house keeping gene) standards, prepare five 1.5 ml microcentrifuge tubes and label them 1, 2, 3, 4, 5. Add 9 μl RNase free H<sub>2</sub>O in all five tubes. Add 1 μl of cDNA (use any one of Sample cDNA) in #1. Mix well and transfer 1 μl from tube 1 to tube 2 (10-fold dilution). Mix well and transfer 1 μl from tube 2 to tube 3. Mix well and transfer 1 μl from tube 3 to tube 4. Mix well and transfer 1 μl from tube 4 to tube 5.
-	Add 0.25 μl of each dilution to wells corresponding to 18S Standards (5 different dilutions in triplicates; B1-B15).	+	#Add 0.25 μl of each dilution to wells corresponding to 18S Standards (5 different dilutions in triplicates; B1-B15).
	#Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.		#Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.
	#Run in real time PCR machine.		#Run in real time PCR machine.

-		+	*Real Time PCR Machine
-	Real Time PCR Machine	+	# Centrifuge 384-well plate for 5 min at 2000 rpm.
-	1. Centrifuge 384-well plate for 5 min at 2000 rpm.	+	# Use ABI PRISM 7900HT.
-	2. Use ABI PRISM 7900HT.	+	#Click SDS 2.1 icon.
-	3. Click SDS 2.1 icon.	+	#File – New – Absolute Quantification
-	File – New – Absolute Quantification	+	#Add Detector – Select Genes and Click “Copy to Plate Document”
-	Add Detector – Select Genes and Click “Copy to Plate Document”	+	#In Setup pane, select regions of each gene and click “use”
-	In Setup pane, select regions of each gene and click “use”	+	#Task = unknown, Quantity = 0
-	Task = unknown, Quantity = 0	+	#For standards,Task = standard, Quantity = 16, 8, 4, 2, 1, Passive Ref = ROX
-	For standards,	+	#In Instrument pane, Sample Volume = 13 l
-	Task = standard, Quantity = 16, 8, 4, 2, 1	+	#In Real Time pane, click “open/close” to open. Place plate, then click “close”.
-	Passive Ref = ROX	+	#Save Changes – “*.sds”
-		+
-	In Instrument pane, Sample Volume = 13 l	+
-		+
-	In Real Time pane, click “open/close” to open. Place plate, then click “close”.	+
-		+
-	Save Changes – “*.sds”	+

	==Notes==		==Notes==

Christopher Jacobs: /* Solutions */

2008-05-07T21:43:34Z

Solutions

←Older revision		Revision as of 21:43, 7 May 2008
Line 23:		Line 23:
	===Solutions===		===Solutions===
	Reverse Transcription		Reverse Transcription
		+
	Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):		Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):


-	For ~~1 Reaction~~	+	For one reaction

	*MgCl2 8 μL		*MgCl2 8 μL
Line 34:		Line 35:
	*dTTP 0.4 μL x (N+1) for N reactions		*dTTP 0.4 μL x (N+1) for N reactions
	*dCTP 0.4 μL x (N+1) for N reactions		*dCTP 0.4 μL x (N+1) for N reactions
-	*RNase Inhibitor 2 μL	+	*RNase Inhibitor 2 μL
	*Reverse Transcriptase 2 μL		*Reverse Transcriptase 2 μL
	*Random Hexamer 2 μL		*Random Hexamer 2 μL
	*DEPC Treated H2O 0.4 μL		*DEPC Treated H2O 0.4 μL
-
-

	==Procedure==		==Procedure==

Christopher Jacobs at 01:17, 6 May 2008

2008-05-06T01:17:09Z

←Older revision		Revision as of 01:17, 6 May 2008
Line 20:		Line 20:
	* RNase away		* RNase away
	* Marker		* Marker
		+
		+	===Solutions===
		+	Reverse Transcription
		+	Using GeneAmp RNA PCR Core Kit mix the following (keep everything on ice):
		+
		+
		+	For 1 Reaction
		+
		+	*MgCl2 8 μL
		+	*10X PCR Buffer 4 μL
		+	*dGTP 0.4 μL x (N+1) for N reactions
		+	*dATP 0.4 μL x (N+1) for N reactions
		+	*dTTP 0.4 μL x (N+1) for N reactions
		+	*dCTP 0.4 μL x (N+1) for N reactions
		+	*RNase Inhibitor 2 μL
		+	*Reverse Transcriptase 2 μL
		+	*Random Hexamer 2 μL
		+	*DEPC Treated H2O 0.4 μL
		+


	==Procedure==		==Procedure==
-	~~# Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl~~	+	* Reverse Transcription
-	~~## Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)~~	+	#In PCR reaction tube, add 20 μL of Master Mix (from Step 1) and 20 μL of RNA sample (appropriate amount containing 3 μg of RNA and then q.s. to 20 μL). Centrifuge briefly (~10 sec).
-	##In tube #1, ~~put in 120 µl~~ of ~~BSA, in all other tubes put in 60 µl~~ of ~~RIPA lysis Buffer~~ (~~you always want~~ to ~~use the same dilutant material as what you used to isolate your protein~~)	+	#Add 20 μL of liquid wax to tube such that total volume in tube is 60 μL.
-	#~~#Pipette 60 µl~~ of ~~BSA from~~ tube ~~#1 into #2. Pipette up and down a dozen times or until you think it~~ is ~~properly mixed. Then take~~ 60 ~~µl from tube~~ #2 and ~~put it~~ in ~~tube #3 and pipette up and down. Continue~~ this ~~process through tube #~~5 ~~(this will leave tube #~~5 ~~with 120 µl~~)	+	#Set thermal cycler as below and run 1 cycle (in this lab, use water bath instead):
-	~~#Prepare BCA Working Reagent~~	+	* 20ºC 10 min
-	##~~For the total volume of working reagent calculate:~~	+	*37ºC 30 min
-	##(# ~~standards (~~in ~~our case~~ 5) and ~~samples~~ (30 in ~~our case~~)~~)(~~# ~~replicates (~~2~~))*(volume of working solution per sample (200 µl))~~	+	*99ºC 5 min
-	##~~To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie~~. ~~50 ml Reagent A plus~~ 1 ~~ml Reagent B~~)	+	*4ºC 5 min (but set to 1 hr)
-	#~~Prepare your samples~~	+
-	#~~#Make three concentrations~~ of ~~your samples~~ in ~~three new microcentrifuge tubes labeled S1~~, ~~S2, S3(to ensure you get within~~ the ~~range of 0.125~~-~~2 µl~~)	+	##Freeze in -20ºC until further use.
-	#~~##Put 60 µl~~ of ~~your sample~~ in tube S1	+
-	###Make a 1:2 ~~dilution~~ (~~30 µl sample + 30 µl RIPA buffer~~) in S2	+	* Real Time PCR
-	#~~##Make a~~ 1~~:10 dilution~~ ( 10 ~~µl sample + 90 µl RIPA buffer~~) ~~in S3~~	+	# Add the following reagents in a 1.5 ml tube and mix well (do this step for each gene separately in triplicates):
-	~~#Prepare your Microplate~~	+	## Taqman PCR Master Mix 2.5 μl
-	~~##Pipette~~ 25 µl of each ~~standard or unknown sample replicate into the designated microplate well~~	+	## 20X Primer & Probe 0.25 μl x (N+1) Reactions
-	##Add ~~200 µl of the WR~~ to each well ~~and mix plate thoroughly on a plate shaker for 30 seconds~~	+	## RNase free H2O 2 μl
-	#~~#Incubate~~ plate ~~at 37C~~ for ~~30 minutes~~	+	Total Volume 4.75 μl
-	~~##Remove plate~~ and ~~measure the absorbance at 562 nm on a plate reader~~	+
-	~~##Subtract the average 562 nm absorbance measurement~~ of ~~the Blank standard replicates from the 562 absorbance of al the individual standard~~ and unknown	+	#Add 4.75 μl of mixed reagents to each well corresponding to specific genes (in this lab, we are only looking at 18S; the wells are A1-A12 and B1-B15) in the 384-well plate.
-	~~##Prepare a standard curve by plotting the average Blank~~=~~corrected 562 nm measurement for each BSA~~ standard ~~vs. its concentration in µg~~/µl. ~~Use the standard curve to determine the protein concentration of each unknown sample~~	+	#Add 16 μl of RNase free H2O to 2 μl of cDNA (1:9 dilution) in sterile microcentrifuge tube.
		+	#Add 0.25 μl of diluted cDNA in Sample wells (in this case, A1-A12). Make sure the pipetting volume is consistent.
		+	#For 18S (house keeping gene) Standards, prepare five 1.5 ml microcentrifuge tubes and label them 1, 2, 3, 4, 5. Add 9 μl RNase free H2O in all five tubes. Add 1 μl of cDNA (use any one of Sample cDNA) in #1. Mix well and transfer 1 μl from tube 1 to tube 2 (10-fold dilution). Mix well and transfer 1 μl from tube 2 to tube 3. Mix well and transfer 1 μl from tube 3 to tube 4. Mix well and transfer 1 μl from tube 4 to tube 5.
		+	Add 0.25 μl of each dilution to wells corresponding to 18S Standards (5 different dilutions in triplicates; B1-B15).
		+	#Add 8 μl light mineral oil to each well. You may need to pipet in reverse mode as mineral oil is viscous.
		+	#Run in real time PCR machine.
		+
		+
		+	Real Time PCR Machine
		+	1. Centrifuge 384-well plate for 5 min at 2000 rpm.
		+	2. Use ABI PRISM 7900HT.
		+	3. Click SDS 2.1 icon.
		+	File – New – Absolute Quantification
		+	Add Detector – Select Genes and Click “Copy to Plate Document”
		+	In Setup pane, select regions of each gene and click “use”
		+	Task = unknown, Quantity = 0
		+	For standards,
		+	Task = standard, Quantity = 16, 8, 4, 2, 1
		+	Passive Ref = ROX
		+
		+	In Instrument pane, Sample Volume = 13 l
		+
		+	In Real Time pane, click “open/close” to open. Place plate, then click “close”.

		+	Save Changes – “*.sds”

	==Notes==		==Notes==
-	Used at Stanford for Tissue Engineering Lab Course (ME385B ~~and 2007 Winter/Summer TC Workshops~~	+	Used at Stanford for Tissue Engineering Lab Course (ME385B)
	<!--		<!--
	#List troubleshooting tips here.		#List troubleshooting tips here.
Line 63:		Line 105:

	==Contact==		==Contact==
-	*Originally prepared by CRJ-EJC 1/3/06	+	*Originally prepared by CRJ-EJC 3/1/04

Christopher Jacobs at 00:57, 6 May 2008

2008-05-06T00:57:16Z

←Older revision		Revision as of 00:57, 6 May 2008
Line 4:		Line 4:

	==Materials==		==Materials==
-	* ~~BCA reagent A~~	+	* GeneAmp RNA PCR Core Kit (Part# N808-0143, Applied Biosystems)
-	* ~~BCA reagent B~~	+	* PCR reaction tube
-	* ~~96 well plate~~	+	* Centrifuge
-	* Microcentrifuge tubes	+	* Liquid Wax
-	* ~~Microcentrifuge tube rack~~	+	* Thermal cycler
-	* ~~Microcentrifuge~~	+	* RNase free Microcentrifuge tubes
-	* ~~BSA stock~~ (~~2 µg/ µl~~)	+	* RNase free H2O
-	* ~~Pipette~~	+	* Taqman PCR Master Mix (Applied Biosystems 4304437)
-	* ~~Pipette~~ tips	+	* 20X Primers and Probes (Applied Biosystems)
		+	* 384-well plate (Applied Biosystems 4309849)
		+	* Optical cover (Applied Biosystems 4311971)
		+	* Light mineral oil (Fisher M5904)
		+	* Real Time PCR machine
		+	* Various pipet tips and pipetter
		+	* RNase away
		+	* Marker

Christopher Jacobs: New page: ==Overview== Protocol for Real-Time PCR ==Materials== * BCA reagent A * BCA reagent B * 96 well plate * Microcentrifuge tubes * Microcentrifuge tube rack * Microcentrifuge * BSA stock (2...

2008-05-06T00:50:15Z

New page: ==Overview== Protocol for Real-Time PCR ==Materials== * BCA reagent A * BCA reagent B * 96 well plate * Microcentrifuge tubes * Microcentrifuge tube rack * Microcentrifuge * BSA stock (2...

New page

==Overview==

Protocol for Real-Time PCR

==Materials==
* BCA reagent A
* BCA reagent B
* 96 well plate
* Microcentrifuge tubes
* Microcentrifuge tube rack
* Microcentrifuge
* BSA stock (2 µg/ µl)
* Pipette
* Pipette tips

==Procedure==
# Prepare Standards ( 2 fold dilutions): 2 µg/ µl, 1 µg/ µl, 0.5 µg/ µl, 0.25 µg/ µl, 0.125 µg/ µl
## Label 5 microcentrifuge tubes 1-5 (1= highest concentration, 5= lowest concentration)
##In tube #1, put in 120 µl of BSA, in all other tubes put in 60 µl of RIPA lysis Buffer (you always want to use the same dilutant material as what you used to isolate your protein)
##Pipette 60 µl of BSA from tube #1 into #2. Pipette up and down a dozen times or until you think it is properly mixed. Then take 60 µl from tube #2 and put it in tube #3 and pipette up and down. Continue this process through tube #5 (this will leave tube #5 with 120 µl)
#Prepare BCA Working Reagent
##For the total volume of working reagent calculate:
##*(# standards (in our case 5) and samples (30 in our case))*(# replicates (2))*(volume of working solution per sample (200 µl))
##To prepare working solution mix 50 parts Reagent A with 1 part Reagent B (ie. 50 ml Reagent A plus 1 ml Reagent B)
#Prepare your samples
##Make three concentrations of your samples in three new microcentrifuge tubes labeled S1, S2, S3(to ensure you get within the range of 0.125-2 µl)
###Put 60 µl of your sample in tube S1
###Make a 1:2 dilution (30 µl sample + 30 µl RIPA buffer) in S2
###Make a 1:10 dilution ( 10 µl sample + 90 µl RIPA buffer) in S3
#Prepare your Microplate
##Pipette 25 µl of each standard or unknown sample replicate into the designated microplate well
##Add 200 µl of the WR to each well and mix plate thoroughly on a plate shaker for 30 seconds
##Incubate plate at 37C for 30 minutes
##Remove plate and measure the absorbance at 562 nm on a plate reader
##Subtract the average 562 nm absorbance measurement of the Blank standard replicates from the 562 absorbance of al the individual standard and unknown
##Prepare a standard curve by plotting the average Blank=corrected 562 nm measurement for each BSA standard vs. its concentration in µg/µl. Use the standard curve to determine the protein concentration of each unknown sample

==Notes==
Used at Stanford for Tissue Engineering Lab Course (ME385B and 2007 Winter/Summer TC Workshops


==References==


==Contact==
*Originally prepared by CRJ-EJC 1/3/06

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


[[Category:Protocol]]
[[Category:Protein]]