Real Time PCR From Cell Culture: Difference between revisions

Line 7:

*SyberGreen PCR Master Mix Applied Biosystems

*96 well qPCR plate

*Primers (Dilute to 5 uM mixture of fwd and rev ~~and then make a working solution of 0~~.4 uL ~~+ 4.6~~ uL ~~water per reaction~~)

*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water)

*Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html

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===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.

#Get 96 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 template per well.

#Add 5 uL primer per well. If using a 384 well plate use 2.5uL

#Add 5 uL primer per well.

#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

#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

@@ Line 7: / Line 7: @@
 *SyberGreen PCR Master Mix Applied Biosystems
 *96 well qPCR plate
-*Primers (Dilute to 5 uM mixture of fwd and rev and then make a working solution of 0.4 uL + 4.6 uL water per reaction)
+*Primers (Dilute to 0.4 uM mixture of fwd and rev.  From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water)
 *Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html
@@ Line 38: / Line 38: @@
 ===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.
-#Get 96 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 template per well.
+#Add 5 uL primer per well.  If using a 384 well plate use 2.5uL
-#Add 5 uL primer per well.
 #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
+#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