BIO RAD DCODE, Руководство пользователя

Страница: 72 / 85

8.5 Staining and Photographing the Gel
DGGE, CDGE, TTGE, and Heteroduplex Gels
1. Remove the gel from the glass plate.
2. Place the gel into a dish containing 250 ml of running buffer and 25 µl of 10 mg/ml
ethidium bromide (50 µg/ml). Stain for 5–15 minutes.
2. After staining, carefully transfer the gel into a dish containing 250 ml of 1x running buffer.
Destain for 5–20 minutes.
3. Place the gel on a UV transilluminator and photograph (Gel Doc ™ 1000 system catalog
number 170-7520 through 170-7527 or Bio-Rad's Polaroid Gel Documentation System,
catalog numbers 170-3742 through 170-3749).
SSCP Gels
1. Remove the gel from the glass plate. For radioactive SSCP gels, proceed to step 5.
Caution: Use caution when handling radioisotopes. Proper handling and disposal of
radioactive material should be followed.
2. Place the gel into a dish containing 250 ml of running buffer and 1:10,000 dilution SYBR ®
Green II (Molecular Probes, Inc.). Stain for about 30 minutes. The gel can also be stained
with Radiant
™ Red stain (Bio-Rad catalog number 170-3122). Place the gel into a dish
containing 250 ml of running buffer and 1:1,000 dilution Radiant Red stain. Stain for
about 30 minutes.
3. After staining, carefully transfer the gel into a dish containing 250 ml of running buffer.
Destain for 30 minutes if needed.
4. Place the gel on a UV transilluminator and photograph.
5. Gels that have been labeled with radioisotopes must be autoradiographed or exposed to
a storage phosphor imaging screen (GS-525 Molecular Imager ™ screen). Carefully place
a 3MM Whatman ® paper on top of the gel. Gently slide your hand across the paper to
adhere the gel to the paper and to remove any air bubbles. Flip the gel over and place a
Saran Wrap
™ plastic wrap evenly on top of the gel without creating any bubbles. This
helps to keep the gel intact and prevents any contamination to the gel dryer. Place the gel
on a gel dryer for about 60 minutes at 60 °C.
6. Expose the gel to film or a phosphor imaging screen. Scan the imaging screen on a storage
phosphor imaging system (GS-525 Molecular Imager ™ system catalog number 170-7320
through 170-8305). Develop the film after proper exposure time.
PTT Gels
1. Remove the gel from the glass plate.
2. To visualize the protein standards, place the gel into a dish containing 250 ml of
Coomassie ® blue stain. Stain for 20–30 minutes.
3. After staining, carefully transfer the gel into a dish containing 250 ml of Coomassie
destaining solution. Destain until the background disappears, usually about 1–3 hours.
4. Gels that have been labeled with radioisotopes must be autoradiographed or exposed to
a storage phosphor imaging screen (GS-525 Molecular Imager screen). Since 35 S or 3 H are
weak beta emitters and are typically used as a radioactive label, the gel should be treated
with a commercial fluorographic enhancing reagent to reduce the film exposure time
(i.e. Amplify
™ from Amersham). Fluorographic reagents are not needed if the sample is
exposed to a phosphor imaging screen.
68