By Jon Lorsch
ISBN-10: 0124201202
ISBN-13: 9780124201200
Laboratory equipment in Enzymology: Protein half B brings jointly a few middle protocols focusing on 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 have now introduced jointly a few middle protocols focusing on protein
Read or Download Laboratory Methods in Enzymology: Protein Part B PDF
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Extra info for Laboratory Methods in Enzymology: Protein Part B
Example text
3 Prepare 100-ml YPAD broth in a 500-ml flask for the transformation. 4 Inoculate 5-ml YPAD media with a colony of yeast using a sterile inoculation loop and sterile technique. Briefly vortex to disperse the cells. Grow the culture overnight at 30 C in a shaking incubator (shaking at 250 rpm). 18 39 The next morning, make a 1:5 or 1:10 dilution of your overnight culture into fresh prewarmed YPAD media. After dilution, there should be enough transferred cells to make the media slightly cloudy.
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.
4. Tip The solution will be very viscous, so you might have to pour the solution into the QIAshredder column. This step reduces the viscosity of the solution by shearing DNA and it also increases RNA recovery. Alternatively, sonication can be used, but this might lead to increased RNA degradation. 62 Emi Sei and Nicholas K. 3 Flowchart of Step 2. 5. Tip Pellet should be minimal after the third spin. In many cases, shorter centrifugation times ($5–10 min) are sufficient. See Fig. 3 for the flowchart of Step 2.
Laboratory Methods in Enzymology: Protein Part B by Jon Lorsch
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