Cas9 Nuclease 
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. The CRISPR system consists of a short non-coding guide RNA (sgRNA) made up of a target complementary CRISPR RNA (crRNA) and an auxiliary transactivating crRNA (tracrRNA). The sgRNA guides the Cas9 endonuclease to a specific genomic locus via base pairing between the crRNA sequence and the target sequence, and cleaves the DNA to create a double-strand break. The location of the break is within the target sequence 3 bases from the NGG PAM (Protospacer Adjacent Motif). The PAM sequence, NGG, must follow the targeted region on the opposite strand of the DNA with respect to the region complementary sgRNA sequence (Fig.1).

Intact Genomics Cas9 Nuclease is the purified recombinant Streptococcus pyogenes Cas9 enzyme containing a nuclear localization signal (NLS) at the C-terminal for targeting to the nucleus. This enzyme is designed to perform CRISPR/Cas9-mediated genome editing. The physical purity of this enzyme is â¥98% as assessed by SDS-PAGE with Coomassie® blue staining.

Quality Control 
Quality control is performed following the production of each new lot of product to ensure that it meets the quality standards and specifications designated for the product. Each lot is repeatedly compared side-by-side with leading competitors to ensure our products outperform the competitor before product launching. 

Cas9 nuclease is free from detectable RNase, Endonuclease (nicking) and non-specific DNase activities.

Product Source 
E. coli BL21 (DE3) strain expressing a Cas9 gene from Streptococcus pyogenes with an N-terminal 6xHis tag and C-terminal SV40 nuclear localization signal (NLS).

Contents & Storage

1. Cas9 Nuclease
2. 10x Cas9 Nuclease Reaction Buffer

Store Cas9 Nuclease and Buffer at -20 °C

Storage Buffer 
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C

1x Cas9 Reaction Buffer 
20 mM HEPES, 100 mM NaCl, 5 mM MgCl, 0.1 mM EDTA, pH 6.5 @ 25 °C

Functional Testing
Cas9 Nuclease functional testing was done by in vitro DNA cleavage assay with the following protocol which gives more than 95% digestion of the substrate DNA as determined by agarose gel electrophoresis.
1) Set up 30 µl reaction in a microcentrifuge tube on ice with the following combinations.

T4 DNA Ligase

Intact Genomics T4 DNA Ligase catalyzes the formation of a phosphodiester bond between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA or RNA.  This enzyme joins DNA fragments with either cohesive or blunt termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids.
 

Quality Control 
Quality control is performed following the production of each new lot of product to ensure that it meets the quality standards and specifications designated for the product. Each lot is repeatedly compared side-by-side with leading competitors to ensure our products outperform the competitor before product launching. 

Intact Genomics T4 DNA Ligase displays up to 3-5X higher ligation efficiency than the nearest competitor.

Product Source
E. coli strain expressing a recombinant clone

Quality Control 
Quality control is performed following the production of each new lot of product to ensure that it meets the quality standards and specifications designated for the product. Each lot is repeatedly compared side-by-side with leading competitors to ensure our products outperform the competitor before product launching. 

Contents & Storage

1. T4 DNA Ligase
2. 10x T4 DNA Ligase Reaction Buffer (w/o ATP)
3. 10 mM ATP

Store all contents at -20 °C.

Storage Buffer 
50 mM Tris-HCl, 50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.5 @ 25 °C

10x T4 DNA Ligase Reaction Buffer (w/o ATP) 
500 mM Tris-HCl, 100 mM MgCl, 100  mM DTT, pH 7.5 @ 25 °C

Note
10x T4 DNA ligase buffer does not contain ATP. You need to add ATP separately.

Unit Definition 
One Weiss unit is defined as the amount of enzyme required to convert 1 nmol of 32P from pyrophosphate into Norit-absorbance material in 20 minutes under standard assay conditions.

Protocol

1. Set up reaction buffer in a microcentrifuge tube on ice. Use a molar ratio of 1:3 vector to insert DNA.

1. Gently mix the reaction and centrifuge briefly.
2. For cohesive ends, incubate 16 °C for overnight or at room temperature for 30 min.
3. For blunt ends, incubate 16 °C for overnight or at room temperature for 2 hrs.
4. Heat inactivate at 70 °C for 15 min.
5. Cool on ice and transform 2 µl of the reaction into 50 µl competent cells.

ig-Fusion Cloning Kit 
Intact Genomics propriety ig-Fusion cloning technology is a simple, rapid and highly efficient cloning kit which allows to directly clone any PCR product(s) to any linearized expression vector at any site. The PCR fragments can be generated by Intact Genomics high fidelity Pfu DNA polymerase or other high-fidelity DNA polymerases, with primers having 15 to 18 bases of homology at their linear ends to where the product need to fuse. The linearized vector can be generated by PCR or restriction enzymes. The kit is so robust that multiple DNA fragments can be assembled simultaneously and cloned into one construct in a single reaction step within short times (usually 10-30 min) with more than 95% cloning efficiency.

Benefits

• Clone any insert at any site within any vector
• Restriction enzyme and phosphatase free system
• Joining multiple large fragments at once
• Precise insertion at a desired orientation
• Rapid and high efficiency with > 95% positive clones

Quality Control 
Quality control is performed following the production of each new lot of product to ensure that it meets the quality standards and specifications designated for the product. Each lot is repeatedly compared side-by-side with leading competitors to ensure our products outperform the competitor before product launching. 

Contents & Storage

• 5x ig-Fusion enzyme premix: -20 °C
• 2x PCR premix: -20 °C
• High efficiency competent cells: -80 °C
• Recovery medium:4 °C or -20 °C

Protocol
1. Linearize the vector by restriction enzyme digestion or inverse PCR and purify the product with spin column.
2. Design PCR primers for the gene of interest with 15 to 20 bp at 5'-extensions that are complementary to the ends of the linearized vector.
3. Amplify the gene of interest with Intact Genomics 2x PCR premix or any other high-fidelity DNA polymerase. Run the PCR product on an agarose gel to determine the integrity of the PCR product.
4. Purify the PCR product with spin column.
5. Set up the ig-Fusion cloning reaction as follows: Insert and vector molar ratio 3:1 produce the highest number of colonies.  

6.  Mix the reaction mixture thoroughly.
7.  Incubate the reaction mixture at 50 °C for 10-30 min, then place on ice. Number of colonies depend on the incubation time, insert size and number of inserts need to clone.
8.  Use 2.0 µl of the reaction mixture and transform into high efficiency ig 10B chemical or electroporation competent cells (included). To get the maximum number of colonies, we recommend to use ig 10B electrocompetent cells (Cat # 1212).

Gel Cutting Tips

• Safely excise bands from gels
• Avoid cross contamination
• One-handed operation for quick and accurate gel cutting
• Safer than using razor blades and won't scratch transilluminators
• 1.1 x 4 mm

This brand is being discontinued.

Need a great DNA Ladders at a great price?

Try our PR1MA™ SmartCheck DNA Ladders!

Bullseye 100bp DNA Ladder RTU (Ready-to-Use)

• A unique combination of PCR products and a number of proprietary plasmids digested with appropriate restriction enzymes to yield 11 fragments, suitable for use as molecular weight standards for agarose gel electrophoresis.
• The DNA includes fragments ranging from 100-1,500 base pairs. The 500 and 1,500 base pair bands have increased intensity to serve as reference points.
• The approximate mass of DNA in each band is provided (0.5 ug a load) for approximating the mass of DNA in comparably intense samples of similar size.

Source:

• PCR products and double-stranded DNA digested with appropriate restriction enzymes, are phenol and equilibrated to 10nM Tris-HCl (pH 8.0) and 10 mM EDTA.

Range: 100-1,500 bp (DL1); 250-10,000 bp (DL5)
Number of bands: 11 (DL1); 13 (DL5)
Concentration: 100 ug / mL
Package: 50 ug / 500 uL
Recommended Load: 5 uL / well
Contains orange G & xylene cyanol FF as tracking dyes. (DL1)
Contains bromophenol blue & xylene cyanol FF as tracking dyes. (DL5)

Storage:

• Store at 25°C for 6 months
• Store at 4°C for 12 months
• Store at -20°C for 24 months

Bullseye 100bp DNA Ladder

• Ready to use
• Contains 11 DNA bands: 100-1500bp.
• Clearly identifiable 500bp band as reference
• 500ng DNA/6 uL/loading
• Easy to load
• Stable at room temperature
• Supplied with 6x sample loading buffer

Bullseye 100bp DNA Ladder consists of 11 DNA fragments ranging in size from 100-1500 base pairs (bp). 6 uL will yield at least 30 ng DNA in any single band. The intensity of the 500bp band has been increased to serve as a reference for easy identification.

Size: 1200 uL
Storage: Store at -20°C.
Concentration: 500 ng/6 uL

Loading Buffer Composition:

• 10mM Tris-HCl
• 1mM EDTA (pH 8.0)
• 0.02% Bromophenol blue
• 0.02% Xylene cyanol
• 5% Glycerol

Usage: Add at least 6 uL Bullseye 100bp DNA Ladder directly to wells designated for markers. You may need more than 6 uL of ladder, depending on well size and level of intensity needed to visualize the bands.

Please give us a call for a sample.

This brand is being discontinued, please contact us for other options.

Need great dNTPs at a great price? Try our PR1MA™ dNTPs 

Bullseye dNTP Mix - 12.5 mM

• Mix of dATP, dCTP, dGTP, dTTP
• Each nucleotide is at a concentration of 12.5 mM
• Ready-to-use molecular grade dNTP solution for use in DNA polymerization, DNA labelling and sequencing processes
• High purity: >98% by HPLC
• Supplied in solution at pH 7.5
• dNTPs are stable at -20°C, avoid multiple freeze/thawing (For long-term usage, aliquoting is recommended)
• Functionally tested with thermostable polymerases

This brand is being discontinued, please contact us for other options.
 

Need great dNTPs at a great price? Try our PR1MA™ dNTPs 
 

Bullseye dNTP Mix - 10 mM 

• Mix of dATP, dCTP, dGTP, dTTP
• Each nucleotide is at a concentration of 10 mM
• Ready-to-use molecular grade dNTP solution for use in DNA polymerization, DNA labelling and sequencing processes
• High purity: >98% by HPLC
• Supplied in solution at pH 7.5
• dNTPs are stable at -20°C, avoid multiple freeze/thawing (For long-term usage, aliquoting is recommended)
• Functionally tested with thermostable polymerases

IBI DEPC-Treated, Nuclease-Free Water is ideally suited for all applications in molecular biology lab including PCR, RT-PCR, restriction enzyme
assays, modifying enzyme assays, transfection, cloning, transformation, and specifically for all RNA assays.

IBI DEPC-Treated Water is manufactured under stringent conditions. The purification process for this product includes continuous
deionization, reverse osmosis, UV-treatment, 0.1µm filtration, followed by steam sterilization in an autoclave. The product is re-autoclaved
to ensure the breakdown of DEPC and release of carbon dioxide that may cause the pH to shift.