QPCR: Difference between revisions
m 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-" |
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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 | #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. | ||