By Magnus Lundgren, Emmanuelle Charpentier, Peter C. Fineran
This quantity provides an inventory of state of the art protocols for the examine of CRISPR-Cas safeguard structures and their functions on the genomic, genetic, biochemical and structural degrees. CRISPR: equipment and Protocols publications readers via strategies which were built particularly for the research of CRISPR-Cas and methods tailored from regular protocols of DNA, RNA and protein biology. Written within the hugely winning Methods in Molecular Biology series layout, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, effectively reproducible laboratory protocols, and tips about troubleshooting and keeping off recognized pitfalls.
Authoritative and cutting-edge,CRISPR: tools and Protocols presents a vast record of instruments and methods to check the interdisciplinary points of the prokaryotic CRISPR-Cas security systems.
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Extra resources for CRISPR: Methods and Protocols
44. Nadja Heidrich et al. and CRISPR RNA stability. RNA Biol 10: 700–707 Kroger C, Colgan A, Srikumar S, Handler K, Sivasankaran SK, Hammarlof DL, Canals R, Grissom JE, Conway T, Hokamp K, Hinton JC (2013) An infection-relevant transcriptomic compendium for Salmonella enterica Serovar Typhimurium. Cell Host Microbe 14:683–695 Jager D, Sharma CM, Thomsen J, Ehlers C, Vogel J, Schmitz RA (2009) Deep sequencing analysis of the Methanosarcina mazei Go1 transcriptome in response to nitrogen availability.
2. Fill top and bottom with 1× TBE:100 mL 10× TBE in 1 L of water. 3. Use the needle and syringe to remove air from the bottom of the gel and to rinse out the wells of the gel (see Note 19). 4. Add 5 µL RNA loading dye to the wells and pre-run the gel at 30 W for 20 min (see Note 20). 5. Rinse out the wells again (see Note 19). 6. Pipet 5–10 µL quenched cleavage reaction into each well. Also run an uncleaved sample (RNA without protein) as a size control. 7. Run the gel for 2 h at 30 W or until the fast dye (bromophenol blue, the darker dye) reaches the bottom of the gel (see Note 21).
After decanting the wash buffer the obtained IB pellet can be stored for a few days at −20 °C (see Note 4). 2 Solubilization of Inclusion Bodies 1. To obtain a Cas protein concentration of ~5 mg/mL, add 5 mL fresh solubilization buffer to 500 mg IB pellet (see Note 5). Resuspend the pellet by pipetting the suspension up and down for a few minutes and then incubate for 3 h at 25 °C on rotary shaker. Typically, at the beginning there are small pellet clumps visible, which should be completely resuspended after 1–2 h of incubation.
CRISPR: Methods and Protocols by Magnus Lundgren, Emmanuelle Charpentier, Peter C. Fineran