Changes

Jump to: navigation, search

Real Time PCR From Cell Culture

140 bytes added, 00:42, 29 March 2011
added reference for calculating amounts from Ct
*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
 
==Calculations==
see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations
==References (Saltiel Lab)==
Retrieved from "http://bridgeslab.sph.umich.edu/protocols/index.php/Special:MobileDiff/548...568"

Navigation menu