Lysis of colonies and DNA binding assay to filter membranes
Lysis of colonies and DNA binding assay to filter membranes
This protocol describes how to release DNA from a clone carrying a recombinant plasmid and immobilize it on a nitrocellulose filter or nylon membrane, a method originally used by Grunstein and Hogness. This protocol is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Experiments on lysis of colonies and binding of DNA to filter membranes
Principle
This protocol describes how to release DNA from a clone carrying a recombinant plasmid and immobilize it on a nitrocellulose filter membrane or nylon membrane, a method originally used by Grunstein and Hogness.
Materials and Instruments
Filter membrane with E.coli transformer clone Move I. Materials For more product details, please visit Aladdin Scientific website.
Denaturing solution Neutralizing solution SDS (10% m V) 2X SSPE
Glass or plastic tray Microwave oven Filter paper
1. Buffers and solvents
Denaturing solution, neutralizing solution, SDS (10%, m/V), 2X SSPE.
2. Specialized equipment
(1) Glass or plastic tray for handling filter membranes.
Trays used in restaurants are ideal for handling 25 sheets of filter membranes with a diameter of 9 to 10 cm at a time.
(2) Microwave oven, vacuum drying oven (up to 80°C) or UV cross-linking device.
(3) Whatman 3 MM filter paper.
3. Carriers and Strains
Filter membrane with E.coli transformer clone.
II. METHODS
1. Cut Whatman 3 MM filter paper (or equivalent) into 4 pieces of appropriate size and shape to fit the bottom of 4 glass or plastic trays. To fit the bottom of 4 glass or plastic trays, soak each of the 4 sheets of 3 MM filter paper in one of the 4 liquids listed below:
10% SDS, Denaturing Solution, Neutralizing Solution, 2X SSPE.
2. Pour off excess liquid and roll the filter paper with a 10 ml pipette to remove air bubbles between the paper and the bottom of the tray.
If the filter paper is too wet, the bacterial clones will swell and expand during the lysis process, resulting in blurred and weakened hybridization signals, which makes clone identification based on hybridization signals very difficult.
3. Remove the nitrocellulose membrane or nylon membrane from the agar plate with blunt forceps, and place the colony face up on the SDS-soaked 3 MM filter paper for 3 min.
This treatment prevents diffusion of plasmid DNA during denaturation and neutralization, thus giving a clearer hybridization signal.
4. The first membrane is soaked in SDS solution for 3 min and then transferred to the second membrane wetted with denaturing solution. The rest of the membranes removed from the agar plate are also transferred in the same order, with each membrane soaked in denaturing solution for 5 min.
When transferring the membranes from one tray to another, use the edge of the first tray to remove as much water as possible from the membranes, or the membranes can be transferred to a paper towel first to remove excess water. Avoid allowing liquid to remain on the membrane with bacterial clones.
5. Transfer the membrane to a third 3 MM filter paper that has been soaked in neutralizing solution and soak for 5 min.
Optionally, repeat this step once.
6. Transfer the membrane to the last 3 MM filter paper that has been soaked in 2X SSPE and soak for 5 minutes.
We prefer to fill the tray or bath with a volume of 2X SSPE (as will be used for washing the membranes after hybridization) and then allow the membrane from step 5 to float on the surface of the liquid for a few minutes, after which the membrane will be allowed to sink below the surface of the liquid and remain in the liquid while the container is shaken until the last membrane has been processed in the same way. This serves two purposes: it washes the membranes of the neutralizing solution, and it impregnates the membranes with EDTA to chelate divalent cations (e.g., Mg2+ ), thereby inhibiting any residual DNA-degrading nuclease activity.
7. Dry the membrane using one of the following methods:
For DNA fixation by oven-drying: Place the membrane on dry 3 MM filter paper, colony side up, for at least 30 minutes at room temperature.
For DNA fixation by UV cross-linking: Place the membrane on dry 3 mm filter paper soaked in 2X SSPE solution or as recommended by the vendor.
8. Immobilize DNA on the membrane using one of the following methods:
Bake-drying method: Wrap the membrane with two pieces of 3 mm filter paper at the top and bottom, and bake-dry the membrane at 80℃ for 1~2 hours to fix the DNA.
Excessive drying will make the membrane very fragile. Nitrocellulose membranes that have not been fully neutralized will turn yellow or brown when baked and will break easily, and will also significantly enhance the background formed by non-specific hybridization.
UV Crosslinking: DNA is immobilized using a UV crosslinking device as recommended by the vendor.
9. Hybridize the labeled probe to the DNA immobilized on the membrane.
All membranes that are not used immediately in the hybridization reaction should be added to 3 MM filter paper and wrapped in aluminum foil and stored at room temperature.