Post Electrophoretic Analysis
RNA samples should be free of contaminating particles and DNA, with an A260/A280 ratio of 1.9-2.0.
- For highly expressed genes, load 10µg total of RNA. For trace expression detection, load 10µg of poly A+ RNA. Ethidium may be added to the gel (4 µl of 10 mg/ml per 100ml gel = 0.4µg/ml).
- Visualize the gel on UV and photograph with a phosphorescent ruler.
- To remove formaldehyde and EtBr which may hamper transfer and/or recovery, soak the gel in 3 changes of DI water for 10 minutes each.
- Place a platform slightly larger than the gel in the center of a 9 X 13 X 2" tray. Fill the tray with 10X SSC until the buffer is 0.5 cm below the surface of the platform (An inverted gel mold or a sponge make good platforms. Rinse commercial sponges before first use, to remove surfactants).
- Cut a strip of Whatman 3MM paper to the width of the platform and length of the tray. Lay it over the platform so that the ends rest in the buffer. Roll a pipette over the filter to remove any bubbles beneath.
- Flood the filter paper surface with 10X SSC and lay the gel on the filter paper. Roll the gel with a pipette if needed to remove air bubbles.
- Place strips of plastic wrap on the platform around the gel to prevent buffer from bypassing the gel. Alternatively, lay a piece of plastic wrap over the gel and platform, and cut away the plastic over the gel with a razor blade, leaving a "mask" on the tray.
- Flood the surface of the gel with 10X SSC and carefully overlay the membrane onto the gel (Note: handle membrane with gloves or forceps and only at the corners).
- Cut 3 pieces of Whatman 3MM to the size of the gel. Wet one piece and lay it over the membrane. Roll the paper and membrane with a pipette to remove any bubbles.
- Inspect carefully - any gap between gel and membrane will not only block transfer, it may create a "hot spot" on the blot, making the interpretation more difficult.
- Wet each remaining cut piece of 3MM paper, lay over the stack, and roll to remove bubbles.
- Lay a stack (3-4" high) of paper towels, cut to the size of the gel, over the stack.
- Place a glass plate and a 500g weight on top of the stack. Allow to transfer overnight.
POST TRANSFER PROCESSING
- Disassemble capillary stack down to membrane, mark well positions with indelible pen or pencil before removing membrane from gel.
- Rinse membrane in 5X SSC for 5 minutes at room temperature.
Note: The gel will be flattened to approximately 2mm thickness. The gel can be stained with EtBr and checked under UV to determine extent of transfer.
- Cross-link RNA to membrane: Nylon - expose to UV (approx. 150 mj/cm2). Nitrocellulose - Bake at 80°C for 120 minutes.
- (Nylon filter only) Wash filter in 1X SSC + 0.1% SDS at 65° C for 1 hour. This will substantially reduce the background.
- Perform probe purification.
- Prehybridize in hybridization solution (see below) for 1 hour at 65°C, then replace with fresh solution containing probe for hybridization.
NOTE: Many different protocols exist for hybridization reactions and each must be optimized for any given probe. General guidelines are given below.Hybridization solution (filter sterilize and store at -20°C once mixed):
- 5X Denhardt's Solution (see below)
- 100mg/ml Salmon or Herring Sperm DNA
- 0.1% SDS
- 5X SSPE (see below)
- 50% formamide
- 3M NaCl
- 0.2M sodium phosphate, pH 7.4
- 25mM EDTA
- Select a hybridization temperature which will allow annealing of the probe, but prevent non-specific binding to nontarget sequences. Note that high stringency washing later in the protocol may not be able to compensate for too low a hybridization temperature. The result will be a large number of false positive bands.Calculation of theoretical melting temperature (Tm):TmDNA:RNA = 79.8°C + 18.5 (log10[Na]) + 0.58 (%GC) + 11.8 (%GC)2 - 0.50 (%F) - (820/length)Notes:
- Tm decreases by approximately 1° C for every 1% increase in mismatches.
- Tm decreases by 0.5° C for every increase of 1% in formamide (%F).
- A good hybridization temperature to begin with is 20°C below the calculated Tm.
- Washes should be carried out at approximately 15°C below the calculated Tm.
After hybridization, non-specifically bound probe is removed by washing in low salt buffer at high temperature. The salt concentration and temperature must be optimized for each probe/sample combination. A good starting point is to wash one time for 20 minutes in 1X SSC and 0.1% SDS at 45° C, followed by three 20 minute washes in 0.2X SSC and 0.1 % SDS at 65° C.
Peform autoradiography as described previously on the following page.
After autoradiography, the probe may be stripped from the blot, allowing the blot to be re-probed up to 5 times (nylon) or 2 - 3 times (Nitrocellulose). Incubate the blot in 50% formamide, 6X SSC at 65° for 30 - 60 minutes. Wrap stripped blot in plastic wrap and place on film overnight to confirm probe removal. Note: If blot is allowed to dry with probe bound to it, the probe will become permanently attached.
NEXT TOPIC: Southern Blotting
- UV Shadowing
- Uneven Staining
- Staining Proteins Immobilized on Membranes
- Staining Protein Gels with Coomassie Blue
- Southern Blotting
- Smeared Bands
- Silver Staining Protein Gels
- Silver Staining DNA Gels
- Protein Fixation on Gels
- Post-Electrophoretic Visualization with Nuclistain
- Overview of Western Blotting
- Northern Blotting
- Method for Western Blotting
- Mechanism of Immunostaining
- Mechanism of Immunostaining
- Immunostaining with Alkaline Phosphatase
- Guide Strip Technique
- Faint bands, low background
- Faint Bands, High Background
- Ethidium Bromide Staining
- Enzyme Linked Immunosorbent Assay (ELISA)
- Coomassie Blue Stain- Troubleshooting
- Blotches on Gel
- Autoradiographic Enhancement with Autofluor
- An Overview of Northern and Southern Blotting
- Alkaline Blotting
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents