reverse transcription reaction
• Add 5 microliters RNA to a sterile siliconized eppendorf tube along with 1 microliter of oligo dT (0.25 mg/ml).
• Heat to 65° for four minutes and immediately transfer to ice.
• Quickly spin the RNA/oligo dT and add 14 microliters of the RT cocktail:
2 microliters 10X RT buffer
1 microliter 1 mg/ml BSA
1 microliter 20 mM DTT
0.5 microliters RNaseIN
0.4 microliters 25 mM dNTPs
0.2 microliters AMV RT
9 microliters Q
• Incubate at 48° for 30 minutes, dilute 5 fold and store at -20°.
pcr reaction
• Dilute cDNA (5 fold) and use between 1-5 microliters of cDNA per PCR reaction.
• Add the following PCR cocktail per tube:
2.5 microliters 10X PCR buffer
1.5 microliters 25 mM MgCl2
1 microliter each primer (10pmol/microliter; 55° Tm)
0.2 microliters 25 mM dNTPs
0.025 microliters a-32P dATP
0.02 microliters Taq polymerase (protocol T.2)
14 microliters Q
• Perform PCR using the following cycle parameters:
94° for 4 minutes, (94° for 30 seconds, 55° for 30 seconds, 72° for 30 seconds) x n, 72° for 5 minutes.
• The number of cycle should be determined empirically to find the linear range of amplification.
