Real Time PCR From Cell Culture: Difference between revisions

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 ===RT-PCR Reaction===
-#Use 8 uL of RNA per RT reaction.
+#Book PCR Machine for 2h
-#Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
+#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water
-#Store cDNA at -20 until use
+#Combine the following:
-#Typically dilute 5X in water (adjust for higher or lower expressing transcripts)
+##8 uL RNA
+##1 uL dNTP mix
+##1 uL Random Hexamers
+#Put in PCR machine and run program '''RT 65C''' (takes 5 min)
+#In a separate tube add in the following order (for 10 reactions):
+##20 uL 10X RT buffer
+##40 uL 25 mM MgCl2
+##20 uL 0.1M DTT
+##10 uL RNAse Out
+#Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge
+#Sit on the bench for 2 min
+#Add 1 uL SuperScript II RT to each tube
+#Put in PCR machine and run program '''RT Reaction'''.  After run (75 min) place on ice.
+#Add 1 uL RNase H and put in PCR machine and run program '''RT 37'''.  Takes 20 min.
+#Store cDNA at -20 until use.
 ===Plate Preparation===