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.
Figure 3. RT-PCR of Small RNA Library.
• Concatamerization
Set up a Ban I digest of PCR products - 4 hrs incubation at 37℃
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
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, l℃ate 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 65℃. 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 5000g 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 –20℃ with glycogen for >2 hrs.
Figure 5. Agarose Gel Resolution of Concatamerized DNA Fragments
