Background on the art of getting beautiful Southerns:
There are several things to consider. (1) Digesting enough DNA. (2) The DNA must be digested to completion; i.e., no partial digests because they make interpretation difficult if not impossible. (3) The resolution of fragments. This is determined by both the thickness of the gel comb and the speed at which the gel is run.
For Arabidopsis use about 2μg of total DNA; for other plants; e.g., alfalfa, tomato, potato and soybean use 10μg of total DNA. To avoid partial digests the DNA should be relatively clean. Performing the digestion in a very large volume (150μl) with an excess of enzyme also reduces partial digests because it dilutes salts and other potential contaminants. A 1mm thick comb and an overnight run at 30 to 35 V for 14 to 16 hours using TEA gel buffer (TAE) produces highly resolved bands in the size range of about 500bp to about 10kb. To do it this way however requires a period of several days:
Timetable:
Day 1: Digest the DNA
Ethanol-precipitate the digested DNA
Resuspended the DNA in a very small volume (~15μl) of TE overnight
Day 2: Pour and load the gel
Day 3: Photograph and blot the gel
(Include a ruler in the photograph)
Day 4: Disassemble the blot
UV crosslink (Using the Stratalinker) and/or bake the membrane
Go directly to pre hybridization or store filter.
A few additional considerations:
An excess number of samples may preclude that gained by the 1mm comb and the need to ethanol precipitate the digested DNA in order to end up with a very small volume (~15-20μl) that will fit into the well. If this is the case do the restriction in 25-30μl, skip the ethanol precipitation step and use a 2mm thick gel well comb. The 3mM thick combs do not provide good resolution and should be avoided. The 2mM combs allow greater loading volume and are a good compromise.
In my (Eric’s) experience gels run overnight using TEA buffer produce more highly resolved bands than do TBE buffered gels. The problem with TAE is that it has a low buffering capacity. As a consequence the buffers will become polarized; I have experienced the upper reservoir hitting pH>10; when this happens the gel begins to melt, which is not good. To get around this either run the gel using a gel apparatus that holds a large volume of buffer and/or with TEA overnight in one of the gel boxes that allows buffer exchange between the cathode (-) and anode ( ) buffer reservoirs.
If many samples will be run that are identical except for one component; e.g., the DNA or the enzyme, prepare a cocktail of all the common components and then aliquot this cocktail out to the different tubes (keeping everything on ice the entire time).
Basic Digest (perform everything on ice):
At the conclusion of the digest either (a) Ethanol precipitate the DNA (which can also be stored at –20℃ to spin at a later date), (b) Load it on the gel (c) Freeze it away at –20oC to be processed later or (d) Add loading dye (if DNA is at the desired volume) and load on gel or freeze at –20℃.
Hint: If you are digesting multiple samples, prepare a cocktail of all components minus the one unique component. For example if you want to digest one particular
DNA with 5 different enzymes, prepare a master mix cocktail that contains everything except the enzyme. Of course this assumes that all enzymes chosen will cut in the same buffer (BSA does not matter; i.e., you may include it without any negative consequences for any enzyme). Dole out the cocktail to the tubes on ice and then add the
DNA. Generally it is wisest to prepare slightly more than needed; i.e., if eight
DNA samples will be digested, then prepare enough master-mix cocktail for 10 samples, etc.
Ethanol precipitation (assuming 150μl volume):
Resuspend the DNA pellet overnight in 15μL (or any convenient volume) of T10E0.1. (With or without loading dye, I don’t think it matters). Some people simply incubate the T10E0.1 and DNA at 60-65℃ ~20 minutes to resuspend the DNA.
Electrophoresis and Blotting
Load the gel.
Run for approximately 500V-h overnight. This is a general rule of thumb. For example 35V for 14h = 490 V-h works great to give beautiful Southern Blots.
Photograph the gel. Include a ruler in the photograph. It is probably easiest if the ruler “0” mark is aligned at the origin (the well). The markings on the gel should be visible in the photograph. If you want you can use one of the several rulers in the lab that fluoresce when put on the uv box. The problem with these is that often there is too much florescence from the ruler.
Determine the dimensions of the gel that need to be blotted. This would be from the origin to the bottom, and the width of the gel. If only a subset of lanes were loaded then those would be the dimensions (plus a little extra).
Place the gel into a container slightly larger than the gel itself. This can be anything from a glass pyrex baking dish to a plastic Rubbermaid container.
Add approximately 250ml of “denaturation solution,” enough to cover the gel and allow movement of the gel solution. Place the gel on a shaker platform and gently shake for about 15 minutes at RT. Decant off the denaturation solution. An easy way to do this is to hold the gel in the tray with a gloved hand and tip the container over the sink.
Add a second 250ml volume of denaturation solution and incubate for approximately another 15minutes. Decant off the denaturation solution.
Rinse the gel by filling the container with tap DI water and decanting off the water. Add approximately 250ml of “neutralization solution.” Return the gel to the shaker platform and gently shake for about 30 minutes at RT. Decant off the neutralization solution.
Add a second 250ml volume of neutralization solution and incubate for approximately another 30 minutes.
Meanwhile, while the gel is incubating in the denaturation/neutralization solutions, prepare the Hybond N transfer membrane (bottom left drawer opposite the –80℃) and the S
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