Tải bản đầy đủ - 0trang
Protocol 3.1 b: Bidirectional Blotting: A Sandwich Blot
SOUTHERN BLOT ANALYSIS
1. Run the gel. Stain and photograph the gel.
2. Soak the gel in wick buffer for 10 to 30 min. Use 10 min for a small
gel; use 30 min for a large gel.
Wick buffer: 0.4 N NaOH; 1.5 N NaC1. Add 16 g NaOH and
87.6 g NaC1 per liter of H20.
3. Cut 3 pieces of thick Whatman filter paper (3omm chromatography
paper) and the nylon membrane to fit the gel.
4. Soak the nylon membrane in H20 to wet it and then soak it in wick
buffer until ready to use.
5. Set up a blotting tray, wick, wick buffer, and gel as for the standard
6. Place a weight on top of the stack of paper towels and blot for 4 to
7. Take off the nylon membrane and wash it for 5 min in 5 • SSC. Bake
the membrane at 60-80~ in a vacuum oven for 60 min.
1. Although this method is more rapid, it can result in poor transfer
of some DNA fragments compared to the standard Southern blotting
protocol. Prolonged transfer times with alkaline blotting can reduce
the DNA bound to the membrane.
2. Recommended prehybridization and hybridization solutions for Nytran
or Gene Screen Plus differ slightly from those for nitrocellulose.
PROTOCOL 3. ld:
This procedure allows DNA from individual bacterial colonies to be
transferred to a membrane. The membrane can then be probed with a
specific DNA to determine if the bacteria contains that sequence. Colony
hybridization is useful to screen many recombinant DNA colonies to determine which colonies contain a specific DNA sequence.
Perform the following steps in advance:
1. Streak bacterial colonies onto the appropriate selective medium. Incubate plates at the appropriate temperature overnight.
2. Use circular nitrocellulose filters that just fit inside the petri plates.
Place the nitrocellulose filters between layers of filter paper. Wrap the
filters in aluminum foil and autoclave them on slow exhaust to sterilize.
PROTOCOL 3.1d: COLONY HYBRIDIZATION
3. Just before the transfer process, prepare the following:
A. 20 ml of lysozyme (1.5 mg/ml) dissolved in 25% sucrose, 50 mM
Tris, and 10 mM EDTA, pH 8.0
B. 20 ml of 0.5 N NaOH, 0.1% SDS
C. 20 ml of 0.5 N NaOH
D. 20 ml of 1 M Tris, pH 7.5
E. 20 ml of 0.15 M NaC1 and 0.1 M Tris, pH 7.5
Prepare five petri plates with Whatman filter papers: Place three circular
Whatman filter papers (that just fit the petri plates used) inside a petri
plate lid. Label each petri plate lid and fill each plate with about 5 ml
of one solution (A-E above). The filter papers should be completely
wet but not have much extra solution above the papers.
Place the petri plate lid with solution A, the lysozyme in sucrose, Tris,
and EDTA on ice.
Keep the other petri plates lids at room temperature.
To do colony transfers or colony lifts, perform the following steps:
1. Use forceps that are flamed to surface sterilize them to handle nitrocelo
2. Press a nitrocellulose filter onto the surface of a plate with colonies
grown on it. Leave the filter in place momentarily. Gently rub the
surface of the filter with a gloved hand to ensure uniform transfer of
colonies from the plate to the filter.
3. Remove the nitrocellulose filter from the petri plate and place the
filter, colony side up, in the petri plate containing solution A on ice.
Be sure to keep the plate horizontal. Colonies will smear into each
other if the plate is tipped. Incubate for 1 min.
4. Using forceps, pick up the nitrocellulose filter and drag the filter across
the edge of the petri plate lid to remove the excess solution from the
bottom of the filter. Place the filter colony side up in the petri plate
with solution B. Incubate for 1 min.
5. Again using forceps, remove the filter from solution B and place the
filter in solution C for 1 min.
6. Remove the filter from solution C and place the filter in solution D
for 1 min.
7. Remove the filter from solution D and place the filter in solution E
for 1 min.
8. Remove the filter from solution E and place on a piece of filter paper.
Allow the nitrocellulose filter to air-dry completely. Place nitrocellulose filters between pieces of filter paper. If not completely dried
before baking, filters may stick to the paper.
SOUTHERN BLOT ANALYSIS
9. Bake the filter under vacuum at 80~ for 2 to 4 hr.
10. Hybridize the filter per the usual Southern blot hybridization conditions.
1. This protocol is based on modifications of Thayer (1979). For additional
discussion of the use of colony hybridization, see Grunstein and Wallis
(1979) and Grunstein and Hogness (1975).
2. Plates can be streaked with colonies, grown overnight at the appropriate
temperature, and then wrapped with Parafilm and stored at 4~ Plates
with colonies may be kept at least 4 days before the colony transfers
3. Occasionally, some of the bacterial cell debris still on the filters may
come off in the prehybridization solution. Such a solution will be a
yellowish color. If there is a lot of debris from the filters, change the
prehybridization solution before adding the probe.
4. If many filters are to be hybridized with the same DNA probe, those
filters can be stacked on top of each other in the same hybridization
bag and hybridized together.
Isolation of DNA Fragments by Electroelution
This method uses agarose gel electrophoresis for the purification of
DNA fragments. The DNA obtained can readily be used in nick translation
(to label DNA to use as a probe) or ligation (to clone a piece of DNA)
reactions or be cut with additional restriction endonucleases to map restriction sites within the DNA fragment.
The method of electroelution allows the isolation of a particular band
of DNA from an agarose gel. Often a highly pure grade of agarose, such
as Pharmacia NA agarose, is used for electroelution. The more highly
purified grades of agarose have lower amounts of sulfated polysaccharides.
Polysulfonates can copurify with DNA and interfere with subsequent enzymatic reactions. Alternatively, if a high grade of agarose is not used, after
electroelution, it may be necessary to phenol extract the DNA sample
Time required: 1 hr for restriction endonuclease digestion, 1-1.5 hr
for gel electrophoresis and staining of gel, 1-2 hr for electroelution.
PROTOCOL 3.2: ISOLATION OF ONA FRAGMENTS BY ELECTROELUTION
9 High-quality agarose, such as NA agarose from Pharmacia or Gibco/
BRL Ultrapure agarose
9 Plasmid DNA, digested with appropriate restriction endonuclease
9 Apparatus for gel electrophoresis
9 Gel box
9 Power supply
9 Ultraviolet transilluminator
9 Dialysis tubing
9 Isopropanol or ethanol
9 1• TBE (Tris-borate gel buffer)
9 0.5 • TBE (half-strength lX TBE solution)
9 10• TBE buffer: 0.89 M Tris (Trizma base), 0.89 M boric acid, 0.02 M
9 Gel loading dye: gel loading dye, stop mix, or stop buffer: 50% glycerol;
0.7% sodium dodecyl sulfate (SDS, a detergent); 0.1% bromophenol
blue (BPB, a dye)
1. Digest the plasmid DNA with the appropriate restriction enzymes in
a final volume of 100-200/~1.
2. Using Pharmacia NA agarose or BRL Ultrapure agarose, make an 0.8%
gel in 1• TBE gel running buffer. For a mini-gel apparatus, use 3050 ml of agarose solution. Set up the gel with a wide slot comb to
make a wide well.
3. If there is any question about complete restriction digestion of the
DNA, check a small aliquot of the large restriction digestion on a
different mini-gel to be sure there has been complete cutting with the
restriction enzymes. If the cutting was not complete, another aliquot
of restriction enzyme and an extended digestion time can be used.
When it is clear that the restriction digestion is complete, add gel
loading buffer to the sample. Load the rest of the sample into the
broad slot of the mini-gel, and subject the sample to electrophoresis.
CAUTION: Ethidium bromide is toxic and is potentially a mutagen. Wear
gloves and a laboratory coat when handling ethidium bromide. Dispose
of ethidium bromide-containing materials properly.
CAUTION: The UV transilluminator is a strong source of UV light. Protect
eyes and skin from UV light. Wear UV protective eye glasses or goggles
and a UV protective face shield. Wear gloves and a laboratory coat to
protect skin from UV light.