The overall goal of this protocol is to introduce an optimized silver staining method for quick, easy, and low cost detection of SSR markers in non-denaturing polyacrylamide gels. The rapid genotyping of SSR markers can help answer key questions in the genetic research and application field such as genetic diversity analysis and marker assisted selection in molecular breeding programs. The main advantages of this technique is that it's easy to carry out, takes less time and using fewer chemical reagents than other protocols for the detection of SSR markers.
This method avoids the fixing, stopping, and several washing steps found in conventional protocols. And only requires the two main steps of impregnation and development. Clear images can be produced using this protocol as no background noise for the unambiguous detection of SSR banding patterns.
One can screen approximately 800 DNA samples per day using this technique and it has been used successfully in tobacco and flowering Chinese cabbage research. After preparing PCR products and gel solutions according to the text protocol, use detergent in tap water to wash one set of glass plates and spacers followed by a complete rinsing with distilled water. Set the plates in a plate drying rack to air dry.
Disperse one milliliter of Bind-silane solution onto the inner surface of a rectangular glass plate and dry it for five minutes. Then disperse one milliliter of Repel-silane onto the inner surface of a notched glass plate with a swab, and use a tissue paper to wipe off excess solution before allowing the plate to air dry for five minutes. Assemble the glass plates with spacers with the notched plate on top.
And use casting clamps to clamp both sides of the assembly. Next, pour an appropriate amount of 6%non-denaturing polyacrylamide gel solution into a beaker for the plate set. Add 20 microliters of TEMED and 200 microliters of fresh 20%APS and gently mix.
Immediately and carefully pour the solution into the assembled glass plates along the edge of the notched plate, filling the space almost to the top. And insert the comb. Then add a small volume of gel solution over the comb and allow the gel to set at room temperature for 30 minutes.
When the gel is fully polymerized, remove the casting clamps and position the plate set in the electrophoresis tank unit with the notched plate facing toward the upper buffer reservoir. Use large clamps to attach the plate set to the tank unit. Pour one liter of 0.5 X TBE buffer into each of the upper and the lower chambers.
Then remove the comb and use a pipette or syringe filled with buffer to thoroughly flush all the wells. To run the polyacrylamide gel, load about one microliter of PCR product into each well. Also, load a DNA ladder on either end.
Then fix the safety cover to the upper buffer chamber. Connect the leads to the power supply, matching the color coded red to red, and black to black. Run the gel at a constant voltage of 110 volts until the dye reaches a defined position.
Normally, for a approximately 70 minutes. Following electrophoresis, open the valve and drain the buffer from the upper chamber into a large beaker. Detach the side clamps and remove the plate from the apparatus.
Then use a spatula to carefully separate the notched glass plate along one side and make sure that the gel remains attached to the other glass plate coated with Bind-silane. Next, prepare one liter of fresh impregnation solution by dissolving 1.5 grams of silver nitrate in one liter of distilled water. Then prepare one liter of fresh development solution by dissolving 10 grams of sodium hydroxide in 900 milliliters of distilled water.
Add one milliliter of 37%formaldehyde and use distilled water to adjust to a final volume of one liter. With ample distilled water, carefully rinse the gel and glass plate to remove the electrophoresis buffer. Then place the plate with the gel facing up into a plastic tray and submerge the gel in one liter of impregnation solution.
Gently shake the tray on a shaker at 60 RPM for three to four minutes. Transfer the plate out of the impregnation solution into another tray. Then use ample distilled water to rinse the residual impregnation solution off of the surface of the plate and the gel two times for three to five seconds each.
The gel wash step after impregnation is critical. Insufficient washing my cause incomplete removal of impregnation solution. And result in dark background.
Place the place with the gel facing up into another tray. Submerge the plate in one liter of development solution then gently shake the tray at 50 RPM for approximately three minutes. Monitor the appearance of DNA fragments and stop development when the highest ratio of the signal of DNA fragments to the noise of background is observed.
The appropriate developing time is another key step. Overdevelopment can result in a dark brown background with a low contrast image of DNA fragments. Rinse the plate and the gel with ample distilled water twice and use tissue paper to dry the gel plate.
Then use a scanner and the appropriate software to scan the gel at 300 DPI and adjust the brightness and contrast to get a clear visualization of the DNA bands. Finally, score the banding pattern of the SSR markers based on the DNA fragment sizes in the image. The PCR amplicons presented here were produced using the corresponding SSR primer pairs in flowering Chinese cabbage and tobacco.
After electrophoresis, the polyacrylamide gels were stained using the silver staining protocol demonstrated in this video. Which unambiguously detected the banding patterns of SSR markers. To compare the detection efficiency of different silver staining protocols, PCR products of SSR markers were separated using PAGE, and visualized using five published silver staining protocols and a sixth method demonstrated in this video.
The protocol demonstrated here had the lowest background noise and highest contrast of DNA fragments such that it produced the highest picture clarity. Of the six protocols tested, the method demonstrated here takes the least amount of time and requires the fewest number of chemical reagents and process steps. Finally, this figure shows the sensitivity of the protocol as measured using a serial dilution of a 50 to 2000 bp DNA marker on a non-denaturing polyacrylamide gel from 10 nanograms per band in lane one to 9.8 picograms per band of DNA in lane 11.
Once mastered, this technique can be done in seven minutes if it is performed properly. While attempting this procedure, it's important to remember to prevent cross-contamination of impregnation and development solutions. After it's development, this technique paved the way for researchers to quickly genotype SSR markers.
After watching this video, you should have a good understanding of how to simply and efficient detect SSR markers in a non-denaturing polyacrylamide gel using silver staining. Don't forget that working with chemical reagents can be extremely hazardous and precautions such as gloves, should always be worn while performing this procedure.
