By John T. Belisle, Spencer B. Mahaffey BSc, Preston J. Hill BSc (auth.), Tanya Parish, Amanda Claire Brown (eds.)
Due to the emerging danger of tuberculosis and different mycobacterial infections, tips on how to research the biology of the mycobacteria and to enhance diagnostic, healing and preventative reagents are nonetheless a great deal in want. Mycobacteria Protocols, moment Edition updates and refines the tools of the well-received first variation whereas including newly built equipment culled from the main state of the art examine within the box. starting from the fundamentals of sub-cellular fractionation to complex equipment utilizing really expert development stipulations and entire genome, transcriptome and proteome research, the chapters agree to the Methods in Molecular Biology™ series layout, offering step by step laboratory protocols, lists of helpful fabrics and reagents, and tips about troubleshooting and averting identified pitfalls.
Comprehensive and updated, Mycobacteria Protocols, moment Edition is the suitable source to advertise and stimulate additional examine into those exciting, very important and so much fractious of bacteria.
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Extra info for Mycobacteria Protocols: Second Edition
Centrifuge at 27,000 Â g at room temperature for 15 min. 26. Carefully remove the aqueous layer (upper phase) into the tubes containing the cell debris. 27. Keep the first detergent phase at 48C. 28. Add Triton X-114 to the aqueous layer with the cell debris to achieve a final concentration of 8% (v/v). 29. Gently rock the tubes at 48C for 2 h. 30. Repeat the detergent extraction twice as above described (steps 21 to 29). 31. After the third extraction, keep the pellet (cell debris) to further perform the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex purification.
Add 500 mL RPE, centrifuge for 15 s, discard flow-through. Add 500 mL additional RPE and centrifuge for 2 min. Discard flow-through and spin for 1 min to dry completely (see Note 13). 8. 5-mL collection tube, elute with 40 mL RNase-free water, centrifuge 1 min (see Note 14) and recover eluate. 4 RNA Quantification EC 24 1. Dilute 1 mL RNA sample in 74 mL TE and measure absorbance at 260 and 280 nm to determine RNA concentration and purity (see Note 15). 2. Using the RNA concentrations estimated by the spectrophotometer reading, run 1 mg RNA on a 2% w/v agarose gel.
Mix by vortexing briefly. 3. Transfer mixture to an RNeasy spin column, centrifuge for 15 s, and transfer column to a new 2-mL collection tube. 4. Add 350 mL buffer RW1 (see Note 11), centrifuge for 15 s, and discard flow through. 5. Add 70 mL buffer RDD to 10-mL aliquot of DNase I stock solution and pipette directly onto the column membrane (see Note 11). Allow digestion to continue at room temperature for 15 min to 1 h (see Note 12). 6. Add 350 mL buffer RW1, centrifuge for 15 s. 7. Add 500 mL RPE, centrifuge for 15 s, discard flow-through.