Changes

*SyberGreen SYBR Green PCR Master Mix Applied Biosystems (ThermoFisher Catalog # 4367659; [https://www.thermofisher.com/order/catalog/product/4367659 Vendor Link])

*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]]

#Book 2h on qPCR machine by going to httpsigning up on the sheet in room 7013 and on the [https://slimprimpluscalendar.uthscgoogle.educom/SlimPrimcalendar/index.phpembed?actionsrc=workflow&module_id=4umich.edu_jkmcg58uidmngdh0sutt1d36og%40group.calendar.google.com&subctz=lc480logbookrequestAmerica%2FDetroit google calendar]

#Sketch out the plate in your notes. Typically rows are different primers while columns are different cDNA;'s

#Prepare a Primer/SYBR Green mixture for each primer. For each well you will need 10 5 uL SYBR green + 2.5 uL Primer working stock, so if you have calculated you need 10 wells per primer that is 100 50 uL SYBR Green + 50 25 uL Primers. Make up 20-25% more than you need.#Using the repeater multichannel pipettor pipette put on 2 or 3 tips (depending on your plate arrangement) and set to suck up 122 uL aspirate however many samples you have and dispense 15 7.5 uL per well. #Dispense 15 7.5 uL of Primer/SYBR mixture into each well, dispensing at the bottom of the well.#Using the snap clip ClipTip multichannel add 2.5 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 You don't need to change tipsbetween 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. 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 4Cuntil the machine is ready.#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.

#Open * Set up run using [[Set up qPCR Run on Thermo Cloud and QuantStudio|Thermo Cloud/QuantStudio]] or [[Set up qPCR Run on Roche LightCycler software.|Roche LightCycler]]#Click new experiment.Briefly, log into thermo cloud#Choose SYBR Green option *Select "Design and Analysis New, qPCR" from drop down and make sure that the correct block is in place (it will say 96 or 384).home screen#Click apply template.*Select "set up plate"#Go to templates>run templates> bridges *Select "comparative CT-SYBR Green 384."#Load plate and click 'start run'*Correct volume to read, "10.0uL" instead of "20.0uL"#This protocol is *Select "Plate set up"*Navigate to do the following:drop down menu marked "passive reference" and select "none."#Activate- 1 cycle @95 degrees for 10s*Then import [https://docs.#Amplifygoogle.com/spreadsheets/d/e/2PACX-1vQpOEYWkqKe6tELqIerA7fcwe2LoEQy_8ANGuhOppmGXbD0sBGvKXHnR3z7aYnfEF7- 45 cycles @95, 60 and 73 degrees for 15s, 15s and 10s, respectively-r4FbOd6yCYM/pub?output=csv plate setup] with linked CSV (MUST be a csv file).#Melt- 1 cycle @95, 40, 65, 95 *Save to the cloud and 40 degrees for 1m, 1m, 1s, continuous and 10s, respectivelyupload on the machine when you insert the plate.

[[Category:TranscriptionMolecular Biology ]][[Category:ExpressionRNA ]][[Category:RNAqPCR ]][[Category:qPCRTranscription ]][[ Category: Expression ]]