By Jon Lorsch
Laboratory equipment in Enzymology: Protein half B brings jointly a few middle protocols targeting protein, conscientiously written and edited through experts.
- Indispensable device for the researcher
- Carefully written and edited by means of specialists to comprise step by step protocols
- In this quantity we now have introduced jointly a couple of center protocols focusing on protein
Read Online or Download Laboratory Methods in Enzymology: Protein Part B PDF
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Extra info for Laboratory Methods in Enzymology: Protein Part B
Sample text
Grow cultures overnight at 37 C, with shaking (250 rpm). The next day, isolate the plasmid DNA using a plasmid DNA miniprep kit. Digest the plasmid DNA with the restriction enzymes used to clone the cDNA library into the pPC86 vector. Run the digested plasmid DNAs on an agarose gel to determine the sizes of the inserts (see Agarose Gel Electrophoresis). Send the unique clones for sequencing at a DNA sequencing facility, using primers designed for use with the pPC86 vector. When sequencing results are obtained, compare the sequences of your cDNA clones with all known transcripts of the appropriate organism using the NCBI database.
8 Centrifuge at 2400 Âg at 4 C for 1 min. Remove supernatant. 9 Snap-freeze the pellet on dry ice. Samples can be stored at À80 C indefinitely. 3. Tip Adherent cells should be 70–100% confluent at this point. Be careful not to detach cells from plate. 2. 4. Tip Make sure to level each dish during cross-linking to avoid dry areas. Keep plates on ice at all times. 5. Tip This parameter might need optimization (a reasonable test range is 125– 1500 mJ cmÀ2). Dishes should be kept on ice while UV crosslinking; we place them on an 800 Â800 Pyrex ® tray with ice.
The irreversible covalent bond formed upon UV cross-linking permits the use of high stringency conditions, which UV Cross-Linking of Interacting RNA and Protein in Cultured Cells 55 are necessary to ensure that RNA–protein interactions are not occurring in the lysate. Because live cells are exposed to UV, demonstration of a UV-dependent cross-link is strong evidence that a particular RBP binds a specific RNA in vivo. In addition, since UV irradiation only cross-links closely associated proteins and nucleic acids, positive results indicate a direct RNA–protein interaction.