By Elena, Ed. Hilario
Protocols for Nucleic Acid research via Non-radioactive Probes, moment version offers an organization history at the easy preparative protocols required for the research of nucleic acids through nonradioactive equipment. offering the methodologies utilizing striking new purposes, this quantity deals advisor chapters on nucleic acid extractions, guidance of nucleic acid blots, and labeling of nucleic acids with nonradioactive haptens. New fluorescent concepts comparable to actual Time PCR and microarrays also are incorporated, permitting clients to get a nonradioactive protocol carried out within the laboratory with minimal version required and quickest time to effects. The protocols persist with the profitable equipment in Molecular Biology™ sequence layout, every one providing step by step laboratory directions, an advent outlining the rules in the back of the procedure, lists of the mandatory apparatus and reagents, and pointers on troubleshooting and averting identified pitfalls.
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Additional info for Protocols for Nucleic Acid Analysis by Nonradioactive Probes 2nd Ed (Methods in Molecular Biology Vol 353)
Weigh the pellet and make a 20 mg/mL suspension by adding an appropriate volume of 1X TE. 3. Transfer 100 RL of the 20 mg/mL suspension (2 mg) to a sterile tube. 4. ). 2. 25 mg and 2 mg (see Note 8). 1. 5X TE. They can be stored for short times at room temperature, or long term at –20°C, or indefinitely at –80°C. 2. 25 mg. 5 mg/mL suspension by adding an appropriate volume of 1X TE to the plaque pellet. 3. 25 mg) to a sterile tube. 4. ). 3. Layout Our standard layout includes up to 25 plaque samples, flanked on each side by a set of two DNA standards equal to 105 and 106 cells.
RNA degradation (or contamination) can be detected by: a. A blob at the running edge end of the gel—the RNA is totally degraded. b. A smear with indistinct bands present (this can also mean there is a lot of polysaccharide in the sample). c. The two main ribosomal bands are equal in intensity, or the lower band is higher than the upper band. Extraction of Plant RNA 23 d. There is a bright band at the top of the gel near the loading wells indicating the presence of DNA in the sample. Depending on the use of the RNA, this may not be a problem.
Very slowly add 5 g of ground powder to 15 mL of preheated lysis buffer containing PVPP and freshly added β-mercaptoethanol, and vortex between additions (do not allow powder to thaw or form lumps). 2. Homogenize the suspension using the Polytron homogenizer at maximum speed for 20 s, or until the froth reaches the top of the tube. 3. 25 volumes (4 mL) of cold absolute ethanol to the tube and vortex for 30 s. 4. Put 1 volume of chloroform:isoamyl alcohol into each of two Oakridge tubes and put half of the homogenate into each tube, vortex, and centrifuge at 2000g for 10 min at room temperature.