DNA fragments for DynaMarker® RNA High Probe
Procedure of Northern Hybridization
Agarose gel electrophoresis
Both agarose gels electrophorezed by denaturing gel method and by non-denaturing gel method using DynaMarker® RNA Easy Measurement N can be subjected to northern transfer describes below.
See detail Product Information of DynaMarker® RNA Easy Measurement N (DM170) for electrophoresis procedure. There are several protocols for northern transfer.
In this protocol, a widespread capillary method (upward) is described. Alternatively downward transfer method or electroblotting method may be used.
Treatment of electrophoresised agrose gel
1) After electrophoresis of the agarose gel using DynaMarker® RNA Easy Measurement N, RNA bands can be seen under UV illumination. You can take photographs of gel for reference.
2) Trim away any unused area of the agarose gel (for example, upper area of wells). Soak the gel with RNase-free water for 15 min, two times, with a gentle agitation.
It is not necessary to fragment the RNA or neutralize the agarose gel, which are required for southern transfer.
Preparation of membrane and filter paper
Use gloves and forceps to handle the nylon membrane. Cut a piece of nylon membrane about 1 mm larger than the gel in both dimension. Cut Whatman 3MM filter paper and towel paper to the same dimensions of the gel.
Cut another Whatman 3MM filter paper enough to cover the area of the gel for a bridges between transfer buffer reservoir and filter paper under the gel.
Transfer (See Fig.1)
1) Assemble the transfer apparatus as Fig. 2. Fill the glass dish with enough 20 × SSC.
2) Wet a bridge of filter paper with 20 × SSC, place it on a solid support in the glass dish.
3) Wet 2 -3 pieces of gel-size filter paper with 20 × SSC, place it on the bridge of filter paper.
4) Place the gel on the filter paper. Try to avoid getting air bubbles between the gel and filter paper.
5) Float the nylon membrane on the surface of distilled water and submerge in the water to be wet completely.
6) Surround the sides of gel with strips of Parafilm to prevent the two layers of filter paper from coming in contact with each other.
7) Place the wet nylon membrane on the top of the gel. Make sure that there are no air bubbles between the gel and nylon membrane.
8) Wet 2-3 pieces of gel-size filter paper with 20 × SSC, place it on the nylon membrane.
9) Put gel-size paper towels on top of the filte paper to a height of 5-8 cm, and add a weight (about 500 g) to hold everything in place.
10) Allow transfer requires approximately 2-3 hrs but may be carried out overnight. As the paper towels become wet, they should be replaced.
11) After transfer remove paper towels and filter paper. For orientation purposes, cut a small notch in the upper right hand corner of the membrane and remove membrane from gel.
12) Wash membrane in 5 × SSC for 5 min after transfer.
13) Bake membrane at 65 - 80 ℃ for 30 minutes - 1 hr or until completely dry (A vacuum oven is not necessary when baking nylon membrane).
Alternatively, exposure the nylon membrane to a UV source (254 nm). Total exposure should be approximately 1.6 KJ/m2 for wet membranes and 160 J/m2 for dry membrane.
14) Store membrane desiccated at 4 ℃ until hybridization, the blot is stable at 4 ℃ for several months.
[Reference] Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY