QPCR - Revision history

Davebrid1: Formatting for GCal link

2026-03-23T20:08:13Z

Formatting for GCal link

← Older revision		Revision as of 20:08, 23 March 2026
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	===Plate Preparation===		===Plate Preparation===
	#Book 2h on qPCR machine by signing up ~~on the sheet in room 7013 and~~ on the [https://calendar.google.com/calendar/u/0?cid=Y19jN2RhYmJlMjJiNmQ1YTY0YjAzYzU1NzVmOGUxNmEyZmZjNjAwOTJjYWIyZmYwM2VjY2M1NjkyNzljYmQwY2QzQGdyb3VwLmNhbGVuZGFyLmdvb2dsZS5jb20]		#Book 2h on qPCR machine by signing up on the [https://calendar.google.com/calendar/u/0?cid=Y19jN2RhYmJlMjJiNmQ1YTY0YjAzYzU1NzVmOGUxNmEyZmZjNjAwOTJjYWIyZmYwM2VjY2M1NjkyNzljYmQwY2QzQGdyb3VwLmNhbGVuZGFyLmdvb2dsZS5jb20 Google Calendar]
	#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.		#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
	#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.		#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.

Davebrid1: Updated calendar link

2026-03-23T20:05:39Z

Updated calendar link

← Older revision		Revision as of 20:05, 23 March 2026
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	===Plate Preparation===		===Plate Preparation===
	#Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the [https://calendar.google.com/calendar/~~embed~~?~~src~~=~~umich.edu_jkmcg58uidmngdh0sutt1d36og%40group.calendar.google.com&ctz=America%2FDetroit google calendar~~]		#Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the [https://calendar.google.com/calendar/u/0?cid=Y19jN2RhYmJlMjJiNmQ1YTY0YjAzYzU1NzVmOGUxNmEyZmZjNjAwOTJjYWIyZmYwM2VjY2M1NjkyNzljYmQwY2QzQGdyb3VwLmNhbGVuZGFyLmdvb2dsZS5jb20]
	#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.		#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
	#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.		#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.

Treycarr: Detailed instructions on applying cover, primer storage, slightly increased amount of stock to prepare.

2023-11-08T19:13:39Z

Detailed instructions on applying cover, primer storage, slightly increased amount of stock to prepare.

← Older revision		Revision as of 19:13, 8 November 2023
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	#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's		#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
	#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.		#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
	#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.		#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 20-25% more than you need.
	#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.		#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
	#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.		#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
	#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.		#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
	#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.		#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal. Apply cover by starting in the middle of plate, working out towards edges, and finish by applying high pressure to edges, ensuring a proper seal.
	#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.		#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
	#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.		#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

Mollyec at 19:18, 5 April 2022

2022-04-05T19:18:35Z

← Older revision		Revision as of 19:18, 5 April 2022
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	*Select "Plate set up"		*Select "Plate set up"
	*Navigate to the drop down menu marked "passive reference" and select "none."		*Navigate to the drop down menu marked "passive reference" and select "none."
	*Then import plate setup with linked CSV (MUST be a csv file).		*Then import [https://docs.google.com/spreadsheets/d/e/2PACX-1vQpOEYWkqKe6tELqIerA7fcwe2LoEQy_8ANGuhOppmGXbD0sBGvKXHnR3z7aYnfEF7--r4FbOd6yCYM/pub?output=csv plate setup] with linked CSV (MUST be a csv file).
	*Save to the cloud and upload on the machine when you insert the plate.		*Save to the cloud and upload on the machine when you insert the plate.

Mollyec at 19:15, 5 April 2022

2022-04-05T19:15:40Z

← Older revision		Revision as of 19:15, 5 April 2022
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	===Run Protocol===		===Run Protocol===
	* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio\|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler\|Roche LightCycler]]		* Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio\|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler\|Roche LightCycler]]
			Briefly, log into thermo cloud
			*Select "Design and Analysis New, qPCR" from the home screen
			*Select "set up plate"
			*Select "comparative CT-SYBR"
			*Correct volume to read, "10.0uL" instead of "20.0uL"
			*Select "Plate set up"
			*Navigate to the drop down menu marked "passive reference" and select "none."
			*Then import plate setup with linked CSV (MUST be a csv file).
			*Save to the cloud and upload on the machine when you insert the plate.

	==Calculations==		==Calculations==

Snyderds: added link to google calendar

2019-02-19T20:25:51Z

added link to google calendar

← Older revision		Revision as of 20:25, 19 February 2019
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	===Plate Preparation===		===Plate Preparation===
	#Book 2h on qPCR machine by signing up on the sheet in room 7013		#Book 2h on qPCR machine by signing up on the sheet in room 7013 and on the [https://calendar.google.com/calendar/embed?src=umich.edu_jkmcg58uidmngdh0sutt1d36og%40group.calendar.google.com&ctz=America%2FDetroit google calendar]
	#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.		#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
	#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.		#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.

Mollyec: /* Plate Preparation */

2018-10-23T15:43:41Z

Plate Preparation

← Older revision		Revision as of 15:43, 23 October 2018
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	===Plate Preparation===		===Plate Preparation===
	#Book 2h on qPCR machine by signing up on the sheet in room ~~6041~~		#Book 2h on qPCR machine by signing up on the sheet in room 7013
	#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.		#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
	#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.		#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.

Reddj at 16:29, 14 February 2018

2018-02-14T16:29:01Z

← Older revision		Revision as of 16:29, 14 February 2018
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	#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.		#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
	#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.		#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.
	#Using the repeater multichannel ~~pipettor~~ put on 2 or 3 tips (depending on your plate arrangement) and set to ~~suck up~~ however many samples you have and dispense 7.5 uL per well.		#Using the repeater multichannel pipette put on 2 or 3 tips (depending on your plate arrangement) and set to aspirate however many samples you have and dispense 7.5 uL per well.
	#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.		#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
	#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.		#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.

Elhabbal: added how to make the 100 uM stock: "-the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example-"

2017-05-24T20:32:59Z

added how to make the 100 uM stock: "-the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example-"

← Older revision		Revision as of 20:32, 24 May 2017
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	*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])		*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
	*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)		*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
	*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). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]		*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks- the 100uM stock is prepared by adding 227 uL of distilled water to 22.7nmol of a gene as an example- this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

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

Davebrid: Updated with smaller sample volumes

2017-02-22T20:16:18Z

Updated with smaller sample volumes

← Older revision		Revision as of 20:16, 22 February 2017
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	===Materials===		===Materials===
	*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)\|First Strand cDNA Synthesis (AB Kit)]] for details		*cDNA: see [[First_Strand_cDNA_Synthesis_(AB_Kit)\|First Strand cDNA Synthesis (AB Kit)]] for details
	*~~SyberGreen~~ PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])		*SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])
	*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)		*384 well qPCR plate (ThermoFisher Catalog # 4309849) and covers (Catalog # 4360954)
	*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). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]		*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). This primer mix can be stored at -20 in a Working Primers box. Design primers according to [[Primer Design for qPCR]]

	===Plate Preparation===		===Plate Preparation===
	#Book 2h on qPCR machine by ~~going to http://slimprimplus.uthsc.edu/SlimPrim/index.php?action=workflow&module_id=4&sub=lc480logbookrequest~~		#Book 2h on qPCR machine by signing up on the sheet in room 6041
	#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.		#Prepare cDNA and dilute in water in a 96 well plate. Typically a 20x dilution of cDNA leaves enough to be detected.
	#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.		#Get optically clear 384 well plate and keep on paper towel. Do not touch bottom of plate.
	#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA;s		#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA's
	#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.		#Calculate how many samples x how many replicates/per sample (start with 3 or 4 until you are consistent enough technically to decrease). This will be the number of wells need for each primer.
	#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 10 uL SYBR green + 5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is ~~100~~ uL SYBR Green + 50 uL Primers.		#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 50 uL SYBR Green + 25 uL Primers. Make up 10-20% more than you need.
	#Using the repeater multichannel pipettor put on 2 or 3 tips (depending on your plate arrangement) and set to suck up ~~122 uL~~ and dispense 15 uL per well.		#Using the repeater multichannel pipettor put on 2 or 3 tips (depending on your plate arrangement) and set to suck up however many samples you have and dispense 7.5 uL per well.
	#Dispense 15 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.		#Dispense 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.
	#Using the ~~snap clip~~ multichannel add 5 uL of cDNA to each applicable well. The width between tips should be 2 wells, so you should be able to add to columns 1/3/5/7... in one group and rows 2/4/6/8... in another group. If you add the cDNA to the top of the well and the primer mixture to the bottom you dont need to change tips.		#Using the ClipTip multichannel add 2.5 uL of cDNA to each applicable well. You don't need to change tips between wells.
	#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.		#Once the plate is completed, carefully put an optically clear cover on it using the plastic square to ensure the edges are sealed, being very careful not to leave fingerprints on the seal.
	#You can prepare the plate ~3h beforehand, keeping it at 4C		#You can prepare the plate ~3h beforehand, keeping it at 4C until the machine is ready.
	#Immediately before the run spin the plate briefly in the swinging bucket centrifuge.		#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

	===Run Protocol===		===Run Protocol===