1
edit
Changes
QPCR
,Detailed instructions on applying cover, primer storage, slightly increased amount of stock to prepare.
#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.
#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 10-20-25% 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.
#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.
#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.
#Immediately before the run spin the plate briefly (2 mins at 4000 RPM) in the swinging bucket centrifuge.
