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Real Time PCR From Cell Culture

33 bytes added, 21:49, 10 December 2010
altered primer concentration and volumes for 384 well plate
*SyberGreen PCR Master Mix Applied Biosystems
*96 well qPCR plate
*Primers (Dilute to 5 0.4 uM mixture of fwd and rev and then make a working solution of 0. From 100 uM stocks this is 4uL Forward Primer, 4 uL + 4.6 Reverse Primer and 992 uL water per reactionWater)
*Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html
===Plate Preparation===
#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
#Prepare primer mix from 5 uM primer pair stocks. Per reaction add 0.4 uL primers + 4.6 uL water.#Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.#Add 5 uL template per well. If using a 384 well plate use 2.5uL#Add 5 uL primer per well. If using a 384 well plate use 2.5uL
#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
#Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL
#Start qPCR Machine using the machine in the [[qPCR - Lin Lab|Lin Lab]] or the [[qPRC - Saltiel Lab|Saltiel Lab]] machine