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32118 AapCas12b (C2c1) Nuclease

  • 32118 AapCas12b (C2c1) Nuclease.png
  • 32118.png

32118 AapCas12b (C2c1) Nuclease

  • Specifications:
    • 100 pmoL
    • 1,000 pmoL

Article number:

32118


Price:Contact our distributors


隐藏域元素占位

Product overview

Description

AapCas12b (previously known as AapC2c1) is a DNA endonuclease mediated by tracrRNA and crRNA (or sgRNA), which can specifically cleave the dsDNA target in a PAM (TTN)-dependent manner, generating DSBs, and cleave ssDNA target in a PAM-independent manner. In addition, both dsDNA and ssDNA targets can trigger the trans-cleavage activity of AapCasI2b, that is, when Cas12b binds sgRNA and target DNA to form a ternary complex, the trans-cleavage activity of Cas12b will be released to non-specifically cleave ssDNA sequences in the reaction system. AapCas12b is derived from Alicyclobacillus acidophilus. The optimal reaction temperature is 60°C for AapCas12b. Therefore, compared to AacCas12b (#32116), AapCas12b is more suitable for “One-Step” CRISPR-Dx system with the integration of LAMP isothermal amplification (for details, please refer to PMID: 32937062).

 

 

Product Components

Components

32118-01 (100 pmol)

32118-03 (1,000 pmol)

AapCas12b Nuclease (10 μM) a

100 pmol

1,000 pmol

10 × HOLMES Buffer 1

1 mL

1 mL

a.The concentration of AapCas12b Nuclease is 10 μM (10 pmol/μL);
 

 

Storage Conditions

Store at -20℃; transport at ≤0℃.

 

 

Experimental Results

Fig1. Cis-cleavage.

 

Fig2. Trans-cleavage.

 

 

Experiment Procedure

1.Cis-cleavage

Components

Volume 

Final concentration 

10 × HOLMES Buffer 1

2 μL 1 ×

10 μM AapCas12b Nuclease

0.5 μL 250 nM

10 μM sgRNA

0.5 μL 250 nM

1 μM Target DNA

0.5 μL 25 nM

Nuclease-free water

Up to 20 μL  

◆ Target DNA can be ssDNA or dsDNA with PAM sequence in the cis-cleavage system.

◆ If nucleic acid electrophoresis is used to analyze cis-cleavage products, it is recommended to use dsDNA targets because ssDNA targets will be further cleaved by trans-cleavage-activated Cas12b. For a 20 μL cis-cleavage reaction system, the recommended amount of target DNA is 100 ng to 500 ng, and the length of the target DNA fragment is in the range of 300 bp to 3 kb. The amount of Cas enzyme and sgRNA required for different lengths of target DNA needs to be calculated according to the molar amount of target DNA. It is recommended to keep the molar ratio of Cas enzyme:sgRNA:target DNA at 10:10:1 to ensure that the target DNA is cleaved completely.

◆ Incubate at 60°C for 30 min~1 h, inactivate at 85°C for 5 min, then analyze the cis-cleaved products by nucleic acid electrophoresis.

 

2. Trans-cleavage experiment

Components

Volume 

Final concentration 

10 × HOLMES Buffer 1

2 μL 1 ×

10 μM AapCas12b Nuclease

0.05~0.5 μL 25~250 nM

10 μM sgRNA

0.05~0.5 μL 25~250 nM

1 μM Target DNA

0.5~5 μL 25~250 nM

10 μM ssDNA Reporter

0.05~0.5 μL 25~250 nM

Nuclease-free water

Up to 20 μL  

◆ Target DNA can be ssDNA or dsDNA with PAM sequence in the trans-cleavage system.

◆ The amount of each component in the trans-cleavage system can be adjusted according to different experimental purposes. When the amount is small, each component can be diluted first and then added to the system. AapCas12b Nuclease can be diluted with 1×HOLMES Buffer 1, and it must be used immediately after dilution (If you want to store the diluted Cas12 enzyme for a long term, please use Cas12 Dilution Buffer, #32012). sgRNA, Target DNA and ssDNA Reporter can be diluted with Nuclease-free Water, but for extremely low concentrations of Target DNA (such as LOD experiments), it is recommended to dilute with 0.1 % Tween 20 and use low adsorption centrifuge tubes and tips etc.

◆ For the amount of probe used in the trans-cleavage system and the expected reaction time to reach the plateau, please refer to the specific value of transU of each batch of Cas enzyme. For details, please refer to the COA test report provided by Tolo Biotech.

◆ The real-time fluorescent quantitative PCR instrument detects the fluorescent signal from reaction at 60°C, and collects the fluorescent signal every 30 seconds.

FAQ

Q1: What are the common Cas12b enzymes, and how do they differ from the Cas12a family?

A1: Common Cas12b enzymes are generally more thermally stable than the Cas12a family. For example, AacCas12b can work in a temperature range of 37°C to 55°C, with an optimal temperature of 48°C. On the other hand, AapCas12b is even more thermally stable and can function in a temperature range of 37°C to 65°C. Therefore, AapCas12b is better suited for use with LAMP (60°C to 65°C). 

 

Q2: How to design the sgRNA for AapCas12b?

A2: AapCas12b lacks its native CRISPR array, so its original crRNA and tracrRNA sequences are unknown. However, studies have shown that AapCas12b shares 95% sequence identity with AacCas12b and can efficiently utilize sgRNA designed for AacCas12b. The sgRNA for AapCas12b can be designed based on the following sequence:
5′– GUCUAGAGGACAGAAUUUUUCAACGGGUGUGCCAAUGGCCACUUUCCAGGUGGCAAAGCCCGUUGAGCUUCUCAAAUCUGAGAAGUGGCACNNNNNNNNNNNNNNNNNNNN–3′,  

(The underlined portion represents the spacer region that complements the specific target sequence).

 

Q3: How should the ratio of Cas12b enzyme:sgRNA:target DNA be adjusted in the cis-cleavage system?

A3: To ensure complete cleavage of the target dsDNA, it is generally recommended to use a ratio of Cas12b enzyme:sgRNA:target DNA = 10:10:1. If the sgRNA efficiency is low, you can try adjusting the Cas12b enzyme:sgRNA ratio to 1:1.25~2. Using excessive sgRNA can also be detrimental to Cas12b cleavage efficiency. Additionally, if sgRNA is prepared by transcription, it's important to ensure the purity of the transcription product.

 

Q4: Where are the cleavage sites for dsDNA targets by AapCas12b, and what determines these sites?

A3: The cleavage sites for dsDNA targets by Cas12b family members are determined by the PAM sequence (5′-TTN-3′). For example, in the case of AacCas12b, the cleavage site is located around the 17th position on the non-target strand (NTS) and the 24th position on the target strand (TS) relative to the PAM sequence. The cleavage occurs in an alternating fashion and generates a 7-nt 5′ overhang.

 

Q5: Can both dsDNA and ssDNA targets activate the trans-cleavage activity of AapCas12b? Which is more efficient?

A5: Both dsDNA and ssDNA targets can activate the trans-cleavage activity of AapCas12b, similar to Cas12a. However, ssDNA targets typically stimulate the trans-activity of Cas12b more efficiently, while dsDNA targets usually activate the trans-activity of Cas12a more effectively.

 

Q6: Does AapCas12b exhibit sequence preferences for trans-cleavage?

A6: Some studies have reported that Cas12b enzymes show higher cleavage efficiency on PolyA, PolyT, and PolyC sequences compared to PolyG, suggesting that Cas12b proteins may exhibit sequence preferences for ssDNA substrates. The specific preferences would need to be experimentally verified.

Product Instruction

#32118 AapCas12b Nuclease

pdf

620.4KB

References

COA Query

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