MicroRNA and siRNA Cloning Protocol
Bartel Lab Protocol Updated: July 2005
Incubate reverse transcription reaction at 48oC for 1 hour. Next, add 1 µL RNase H and incubate at 37oC for 30 minutes. Do all steps in parallel with the (-)RT control. Remaining RT reaction may be stored long term at -20oC.
Set up 100 µL reactions for the RT(+) and RT(-) samples for PCR.
5 µL of RT reaction 10 µL 10X PCR Buffer 10 µL 10X dNTPs
15 to 25 cycles of PCR (hot start optional)
1 µL 100 µM 17.92 1 µL 100 µM 17.93D 2 µL Taq Polymerase 71 µL dH2O
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94 C – 30 sec 50 C – 30 sec 72 C – 30 sec
Analyze reactions with a 15% denaturing polyacrylamide gel. Take 3 µL from each RT-PCR reaction, add loading dye, heat well before loading, and load onto a pre-run midi-thickness gel. Run using the 10bp ladder to follow bands. Do not use EtBr for staining, because the sensitivity is very weak for these small DNAs. Use the SYBR Gold stain from Molecular Dynamics. You should see a good smear in the size range of small RNAs ligated with linkers. Use filter tips. Two times phenol extract. Two times chloroform extract. Add NaCl to make 0.3M / EtOH precipitate (glycogen optional). Spin down pellet and resuspend the RT(+) reaction in 40 µL.
10X Bartel Lab PCR Buffer 100 mM Tris pH 8.3 500 mM KCl 15 mM MgCl2 0.1% Gelatin
1X dNTPs contain 0.2mM of each dNTP
Amplified small RNA cDNA Library
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