Safety warnings: This procedure uses methanol which is toxic and flammable. Glove must be worn whenever handling methanol
Materials Needed
100X BioRad Fast Blast DNA stain
PVDF membrane
DNA concentration standards
distilled water
Procedure
Before beginning the assay, practice spotting 1 µl of water onto piece of used PVDFmembrane.
ALWAYS WEAR CLEAN GLOVES WHEN HANDLING THE PVDF (membrane)
1. Prepare the membrane.
With a soft lead pencil, draw a series of 1 cm by 1 cm boxes, one for each sample and for each of the three standards.
“Wet” the membrane by dipping it in methanol and leaving it submerged until it is uniformly darkened.
Transfer the membrane to a dish of distilled water and allow it to soak until it no longer repels water
2. Spot the DNA onto the membrane.
The standards will be provided to you by your instructor. The samples are the samples of DNA that you purified from the plant tissue.
For each standard or sample to be tested, carefully pipette 1 microliter into the center of the box. If done properly, it will form a bead at the spot where you pipetted it. This bead will slowly shrink as the liquid soaks into the membrane.
3. Allow the membrane to dry for 5 minutes.
4. Repeat the “wetting” process from step 1.
5. Place the membrane in a dish with enough 100X Fast Blast DNA stain to cover. Shake for 5 minutes.
6. Transfer the membrane to a dish of distilled water, and shake until you no longer see blue color washing off the membrane. You may need to change the water while doing this.
7. Estimate the concentration of DNA in the sample by comparing the intensity of the blue dot from the sample with the intensity of the blue dots of the standards. The amount of DNA in the standards are 0 ng/µl, 10 ng/µl, and 100 ng/µl.
