John M. S. Bartlett.pdf - Bio-Nica.info

PCR-SSCP Analysis of Polymorphism 331
3.2. SSCP Gel Preparation
1. Prepare a 6% nondenaturing polyacrylamide gel with 10% glycerol as following: Long
Ranger gel solution (7.5 mL), glycerol (7.5 mL), 10× TBE (4.5 mL), H 2 O (55.7 mL);
10% APS (0.4 mL), and TEMED (0.04 mL) for a total of 73.64 mL. Add TEMED and
APS right before pouring the gel.
2. Place a few drops of Sigmacote on the inner side of the shorter plate (see Note 3) and
spread evenly with a paper towel. Allow to air dry. Sigmocote forms a tight microscopically
thin film of silicone on glass, which prevents the SSCP gel from sticking to this particular
plate when you separate the two plates after the gel run is done, allowing the gel to remain
smooth on the other plate. Set up the plates with 0.4-mm spacers on the sides and with
a strip of Whatman chromatography paper at the bottom. Clamp three sides of the plates
leaving the top open.
3. Immediately before pouring the gel, add TEMED and APS to the mixture, collect it into a
60-mL syringe, and push the solution into the plates slowly while holding the plates at an
angle (see Note 7). Place either a 64-well or 36-well comb (see Note 8) into the top of the
gel and clamp the top of the plates. The gel will be polymerized in 2 h.
3.3. Dilution and Denaturation of PCR Products (see Note 9)
1. Dilute 5 µL of amplified DNA in 45 µL of 0.1% SDS and 10 mM EDTA.
2. Mix 5 µL of above diluted PCR products with 5 µL of loading buffer. The sample is ready
to load and may be stored at –20°C for 2 wk.
3.4. Loading Samples and Running Gels
1. Before running the gel, denature the diluted PCR samples by heating at 95°C for 2 min
and cool rapidly in an ice bath.
2. Load 2 µL (for 64-well combs) or 5 µL (for 32-well combs) of the diluted PCR products
onto gel. The leftover samples can be reused if they have been stored at –20°C for less
than 2 wk.
3. Electrophoresis is performed by using 0.6× TBE buffer at a constant 30 watts (W) for
4 h at room temperature (see Note 10).
3.5. Drying Gels and Developing Films
1. After migration, the gel is transferred onto a double layer of Whatman Chromatography
paper and covered with plastic wrap avoiding air bubbles.
2. The gel is vacuum dried in a gel dryer at 80°C for 1 h, and then the gel is placed facing
up in a film cassette.
3. Expose gel to an X-ray film (the X-ray film should be put in between the enhancing screen
and the gel) at –80°C for 16 to 20 h (see Note 11).
4. Warm up film or air dry film before developing. Cut a corner of the film before developing,
which will help to figure out the sample orders to load. Develop film using an automatic
film developer or using the dip method.
3.6. SSCP Gel Analysis
SSCP patterns are analyzed upon visual examination. Analysis is facilitated with
knowledge of the gene sequence, which is often available because sequence information
was necessary to derive primers. Inclusion of control samples of known sequence allow
allele typing (see Note 12 and Subheading 1.).