Cas12b (A. acidiphilus) Recombinant

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Catalog #

101626

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Description

Recombinant A. acidiphilus AapCas12b (type V CRISPR-associated protein Cas12b), tag free. AapCas12 belongs to the type V CRISPR effector, CRISPR-Cas12b/C2c1 and is highly thermostable for a wide range of applications.

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Product Data Gallery

4-20% SDS-PAGE Coomassie Staining

AapCas12b Nuclease Activity

Product Info

Storage and Usage

Citations

Species

Alicyclobacillus acidiphilus

Construct

Cas12b (Full Length) (A. acidiphilus)

Host Species/Expression System

E.coli

Purity

75%

Format

Aqueous buffer solution.

Formulation

50 mM sodium phosphate, pH 7.5, 300 mM NaCl, 1 mM DTT, and 10% glycerol

128 kDa

Amino Acids

Full Length

Genbank #

WP_067623834

Storage/Stability

At least 6 months at -80°C.

Assay Conditions

Varying amounts of AapCas12b activity was measured using a CRISPR-based fluorescent reporter assay for optimal results. Target DNA cutting and indiscriminate single stranded DNA collateral cleavage was activated using RNA-guided DNA Binding to Cas12. Emission of fluorescent signal is due to the degradation of ssDNA reporters upon cleavage.

Active Cas12 was thawed on ice while 1X Endonuclease Buffer containing 10 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10 mM MgCl2, and 0.1 mg/ml BSA, guide RNA (custom designed crRNA), ds DNA activator (complementary sequence to crRNA and a PAM sequence specific for Cas enzyme) and FQ-ssDNA substrate (labeled with fluorophore and a quencher) were equilibrated to room temperature. Next three working solutions of Active Cas12 (4X final concentration) guide RNA (4X final concentration) and activator/reporter mix containing ds DNA activator and ssDNA reporter (2X final concentration), were prepared using 1X Endonuclease Buffer. 10 μl of 4X active Cas12 and 10 μl of 4x guide RNA were then preincubated in half the area of a solid black 96-well plate for 10 minutes at room temperature. After preincubation, 20 μl of 2X activator/reporter mix was added to plate and placed on shaking incubator for 1 min. The plate was then sealed and incubated at 37°C for 10-30 minutes. Plate was then equilibrated to room temperature, plate sealer removed and fluorescence read on a microplate reader. Negative control was measured by replacing enzyme working solution with equal volume of assay buffer.

Instructions for Use

Thaw on ice and gently mix prior to use. DO NOT VORTEX. Perform a quick spin before opening. Aliquot into small volumes and flash freeze for long term storage.

Applications

Useful for DNA cleavage, targeted genome editing, the study of enzyme kinetics, screening inhibitors, and selectivity profiling.

Shipping Temperature

-80°C

Warnings

Avoid multiple freeze/thaw cycles.

Scientific Category

CRISPR

Regulatory Status

Research Use Only

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Datasheets/Protocols

101626 Lot 240123

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