Wear gloves and take appropriate safety precautions when handling.Ĥ. Perform the transfer in a fume hood to avoid inhalation. The gel contains formaldehyde, which will diffuse out of the gel during transfer. Prepare a northern blotting set up for Southern blotting (step 5 to step 12 of Southern blotting protocol). Incubate the gel for 10 minutes in 200 ml of 10X SSC to neutralize the NaOH.ģ.
After gel electrophoresis, incubate the gel in 200 ml of RNase free water and then in 200 ml of 0.05 M NaOH for 15 minutes.Ģ. It is always recommended to handle the membranes with gloves.ġ. Northern blotting is generally carried out using positively charged nylon membranes because of their greater strength and easy handling. Since DNA blotting is commonly referred as ‘Southern blotting’, after its inventor E M Southern, the RNA blotting is called ‘northern blotting’. Preserve the blot if not used immediately at room temperature covered in plasticwrap.Īfter separating RNA molecules in a denatured gel, RNA molecules in the gel are transferred to nylon or nitrocellulose membrane by capillary transfer or vacuum blotting. To UV cross link protect the surface of the DNA side of the membrane with plasticwrap and then expose this side to the UV source for 3 minutes.)ġ5.
To bake, first let the blot dry on a sheet of filter paper, then place between sheets of filter papers, and bake it at 80☌.
(UV cross-linking generally gives better results. Fix the DNA to the blot, either by baking or UV cross linking. Remove carefully the membrane from the gel.ġ4. Mark the positions of gel lanes on the membrane using a ball point pen. Turn over the gel and blotting membrane together (gel side on the top) on a filter paper towel. Immediately after transfer, remove the weight, paper towels and filter papers. Place a weight (about 500 g) on top of the plate.ġ3. Place a glass plate on the top of the paper towels. (Transfer efficiency is improved by removing the wet paper towels and replacing them with dry papers at least once during transfer.)ġ1. Place a 15-20 cm stack of dry paper towels on top of the filter paper. Place two precut sheets of Whatmann 3 MM paper in 10X SSC, and place them on the top of the nylon membrane. Remove any air bubbles between the membrane and gel.ĩ. Place the blotting membrane (nylon membrane) on the top of the gel so that it covers the entire surface. This ensures that the transfer buffer (10X SSC) moves only through the gel and not around it.Ĩ. Remove any air bubbles trapped between the gel and the platform by rolling a pipette several times back and forth over the gel.ħ. Set up a Southern transfer as in Fig.4.1.Ħ. Incubate the gel with neutralization solution containing 1.5 M NaCl and 0.5 M Tris-HCl pH 7.5 for 30 minutes.ĥ. (During denaturation the bromophenol blue will regain its colour.)Ĥ. Denature the double stranded DNA in order to create suitable hybridization targets by incubating the gel with denaturation buffer containing 0.5 M NaOH and 1.5 M NaCl for 15-30 minutes with gentle shaking. Depurination is recommended only for fragments larger than 10 kb or otherwise these yield fuzzy bands or smears in the final autoradiograph, presumably because of increased diffusion of DNA during transfer.)ģ. This depurination step should not be too long, since small fragments attach less firmly with the membrane. During this period the colour of the marker dye (bromophenol blue) will change from blue to yellow, indicating that the gel has been completely saturated with the acid. In order to facilitate their transfer, these fragments are reduced in size, either by depurination or UV irradiation. (DNA fragments larger than 10 kb do not transfer to blotting membrane efficiently. Carefully transfer the gel to 0.2 M HCl and shake for 10 minutes at room temperature. Destain the agarose gel in sufficient amount of water with shaking under room temperature.Ģ. The sequences of interest can be identified by hybridization using radioactive or non-radioactive probes and visualized by autoradiography or staining.ġ. The separated fragments in the gel are transferred to a nylon membrane by capillary transfer or vacuum blotting. Then they are run through an agarose gel. Usually, larger fragments of DNA (genomic DNA) are made into conveniently sized fragments by restriction analysis. Blotting is a technique used to identify specific DNA sequences. Southern Blotting of DNA :ĭNA fragments on agarose gels give no indication of function or sequence. In this article we will discuss about the southern, northern and western blotting of DNA.