MicroRNA and siRNA Cloning Protocol
Bartel Lab Protocol Updated: July 2005
Set up a Ban I digest of PCR products - 4 hrs incubation at 37oC
40 µL of RT-PCR products (Pool 2 tubes) 30 µL NEBuffer 4 10 µL Ban I 20U/µL → 0.67 U final 220 µL dH2O
– Ban I digest –undigested
Check 10 µL from digestion on a 15% denaturing polyacrylamide gel. Use 1 µL from the PCR and the 10 bp ladder as markers, then stain the gel with SYBR Gold. See Figure 4. Two times phenol extract. Two times chloroform extract. Add NaCl to make 0.3M and EtOH precipitate (glycogen optional).
Add the following for concatamerization to the entire pellet from the digest:
8 µL dH2O 1 µL 10X T4 Ligase Buffer (USB or NEB brand is fine) 1 µL T4 DNA Ligase
Incubate at room temp for 30 min. Take a mini-gel casting tray for agarose and rinse thoroughly. Prepare a 2% GTG Nusieve Agarose Gel with 1x TAE, pre-stained with EtBr. Load entire concatamerization reaction with glycerol loading dye into a lane, run with 100bp marker. Run a short time, when the ladder can be visualized. See Figure 5 below.
Using the low energy, high wavelength setting on transilluminator, locate smear corresponding >300 bp concatamers and cut out with a clean razor blade. Add 10 volumes gel melting solution (20 mM TrisHCl pH 8, 1 mM EDTA pH 8) and melt for 5 minutes at 65oC. You may need to distribute this to a couple of siliconized tubes.
Add an equal volume of phenol, vortex for 20 seconds, chill on ice for 5 min, then spin at 5000 g for 10 min (4o C). Remove aqueous phase, re-extract with a 1:1 phenol, chloroform mix, and re-extract again finally with just chloroform. Add 0.06 volume of 5M NaCl and 2.5 volume EtOH and precipitate at –20oC with glycogen for >2 hrs.
Suggested region of gel slice for purification of concatamers
700 bp 500
Monomers, dimmers and trimers
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