Two-Dimensional Thin Layer Chromatography (2D-TLC)
1. Resuspend RNA pellet in 5 µl ddH2O. Place at room temperature for 5-15 minutes. Use pipette tip
to wash inside wall of microfuge tube to collect entire sample. Bath sonicate 1 minute and vortex to
solubilize RNA. Briefly spin intermittantly to collect droplets at bottom of tube.
2. Spin in microfuge at top speed for 5 minutes to collect droplets and pellet any insoluble material.
· If a pellet is visible, carefully transfer supernatant to fresh tube.
3. Optional: Add 0.25 µl 3' rNMP mixture. Vortex. Spin briefly to collect all droplets.
· To prepare stock of 3' rNMP mixture, dissolve 100 mg of each 3' NMP in 100 µl of 25 mM NaOH
(pH >4). NMPs are from Sigma: AMP, A9272; CMP, C1133; GMP, G3628; UMP, U1126.
4. Mark origin on chromatography plate using a pencil. Make a triangle () pattern. The origin is 25
mm from each edge in bottom left corner. Be careful. Do not damage surface of plate at origin.
· Use Merck plates, 20 X 20 cm, pre-coated with cellulose, 0.1 mm, no indicator (from VWR).
5. Spot sample on chromatography plate at origin. Do 0.1 - 0.2 µl at a time. Dry with a gentle stream
of filtered air across origin. Wait until spot is completely dry between applications of sample.
Spot should be 2-3 mm, or less, in diameter. Plate may be stored a room temperature.
Origin: 25 mm
from each side