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

74 Bartlett
4. Many modern electrophoresis tanks are equipped with seals or with buffer dams to allow
pouring of the gel or, alternatively, one can tape over the end of the tray with autoclave
tape. Ensure gels are poured on a level surface.
5. Undissolved agarose appears as small lens-shaped flecks in the solution; reheat until the
solution is clear. Take care not to superheat the solution because it may boil over.
6. Modern electrophoresis equipment will not allow circuits to be completed until the tank is
sealed. If you have an older tank without this safety feature, discard it!
7. If the bands appear faint, with a dark background the gel is understained, return to buffer
containing ethidium bromide and repeat. If the bands appear faint with a bright background
the gel is overstained, destain in excess electrophoresis buffer (without ethidium bromide)
for 10 to 20 min and revisualize.
8. Standard incineration is sufficient to decontaminate ethidium bromide.
9. Ultraviolet light produces strand breaks in DNA. Even exposure for short periods (30 s)
can compromise DNA recovery. Ensure recovery of bands as quickly as possible. Where
multiple bands are to be recovered, we have blocked the ultraviolet from parts of the gel
using cardboard under the gel support. Some protocols recommend the addition of 1 mM
guanosine or cytidine to the gel and electrophoresis buffers to protect DNA at the
transillumination stage.
10. Ensure the agarose is completely melted to optimize digestion.
11. Vortexing can shear large DNA fragments, take care.
12. Although recommended incubation times are shorter, we have found extending the
incubation time can improve the yield and ensure complete digestion of the agarose.
13. If the expected DNA concentration is less than 50 ng/µL, we recommend the addition of
5 µg of tRNA to enhance precipitation.
14. We have successfully worked with 4 to 20% acrylamide gels for various applications. Use
of low concentration acrylamide is only possible in our experience where supports for the
gels (Whatman paper) is provided and this limits the use to radioactive applications.
15. Polymerization will commence as soon as these catalysts are added and is temperature and
time dependent, work quickly but carefully.
16. Wells will frequently contain small amounts of acrylamide solution that has notolymerisind
because of contact with air (oxygen inhibits polymerization). Washing the wells prevents
this from sinking to the base of the well and polymerizing giving uneven well depths.
17. The protean system is cooled by circulation of water through a central core. This is helpful
when rapid results are required, but not essential.
18. Acrylamide gels, especially low percentage gels, are extremely fragile and tear easily, and
handling them is an art learned through practice (and much frustration!). Siliconization
of glass plates can aid removal of gels, but in our experience, providing the plates are
clean separation is relatively easy. If your gel sticks to the glass, clean them thoroughly
before use.
19. A common cause of tearing of gels and problems with pouring is dirty glass plates. Before
use, scrub glass plates with concentrated detergent, rinse extensively with tap water and
distilled water, and then with methylated spirits. Dry thoroughly before use. Clean spacers
and combs by wiping with methylated spirits.
20. Modern powerpacks provide the option of running gels at constant temperature (usually
65°C), and we have found this to provide more consistent results.
21. The use of a silicon solution on the backplate after washing can facilitate separation. Be
pragmatic: If the gel sticks to the back plate remove the front plate. When silver staining
is to be used, stain the gel in situ on the plates.
22. If the gel cracks into a “crazy paving” type pattern while drying, this is indicative of poor
fixation. Extend fixation period. This approach can be used with thicker gels (1–1.5 mm),