By Peter G. Isaac
This ebook presents an extremely important selection of attempted and confirmed protocols for nucleic acid research that keep away from using radioisotopes. Southern (i.e., DNA) and northerly (i.e., RNA) blotting protocols are lined intimately, from how one can isolate quality nucleic acids from plant and animal assets, to using the widest diversity of probe platforms (involving digoxigenin, biotin, fluorescein, horseradish peroxidase labeling, and colorimetric and chemiluminescent detection). different concepts taken care of intimately comprise hybridization in situ to chromosomes and transcribed RNA, more moderen and not more traditional applied sciences, resembling Randomly Amplified Polymorphic DNA (RAPD), opposite dot blots, bacterial identity utilizing magnetic bead and dipstick assays, and nucleic acid sequence-based amplification.
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Use the lid as a template and trace around the outside with a permanent marker pen. This can then be cut out with a clean pair of scissors, Cut a window in the mask just smaller (OS cm in each direction) than the size of the gel to be transferred (in this way the gel should overlap the mask yet all the wells should be within the window). Use a clean ruler to measure the dimensions and draw along the ruler with a permanent marker pen. Cut out the window with a clean pair of scissors. 28. Nylon membrane: Biodyne B or similar (see Notes 5 and 6).
This can cause uneven transfer of the DNA. 23. The Millipore Milliblot butterfly nuts can cause damage to the apparatus if tightened too much. Bulldog clips create a better seal and cause less damage. 24. The conical flask collects any excesstransfer solution from the blotting apparatus, preventing it from reaching and posstbly damaging the pump. An in-line filter (such as Millipore Millex FGsa) can be inserted between the conical flask and the pump as a further protective measure. 25. The vacuum pressure is critical; if the pressure is too high the gel will collapse, trapping the DNA in the matrix and leading to inefficient trans- 36 Stacey and Isaac fer; rf the pressure is too low the vacuum may be insufficient to hold the gel in place resulting in the gel rising to the surface of the transfer solution.
P-mercaptoethanol. RNA Extraction from Mammalian Cells 43 3. Antifoam A (30% [v/v]) from Sigma (St. Louis, MO). 4. Sarcosyl30%. 5. 45 pm) and autoclave. Store at 4°C . 6. 1 rotor or similar. 7. 0 with acetic acid. 8. Absolute ethanol (stored at -2OOC in a flashproof freezer). 9. 70% Ethanol (stored at -2OOC in a flashproof freezer). 10. Sterile distilled water (see Note 1). 11. 1. and Note 1 for use in treating solutions). 12. 0, 1 mM EDTA. 13. Phenol equilibrated with TE (see Note 2). 14. 15 mL 30% antifoam A.
Protocols for Nucleic Acid Analysis by Nonradioactive Probes by Peter G. Isaac