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).

RT 2X Master Mix

• Up to 9kb cDNA synthesis
• Ensures sample to sample consistency
• Large RNA sample volume capacity
• Ready to use

Size: 100 rxns

RT 2X Master Mix is a proprietary, ready-to-use master mix for first-strand cDNA synthesis in a 2X concentration. This optimized reaction mix contains ribonuclease inhibitor, dNTPs, and a balanced concentration for oligo(dT) and random primers. The ribonuclease inhibitor effectively protects RNA template from degradation. The oligo(dT) anneals selectively to the poly(A) tail of mRNAs and the random primers do not require the presence of poly(A) and they are utilized for the transcription of mRNA 5-end regions. The resultant cDNA can be directly used as template in different PCR experiments.

Kit Components

Storage

• Store at -20°C in a frost-free freezer.

Application

• cDNA synthesis
• Construction of cDNA libraries
• Generation of probes for hybridization 

Protocol

1. Thaw RNA templates and all reagents on ice. Mix each solution by vortexing.
2. Assemble the following components in a tube on ice, and mix well:

1. Heat the mixture at 65°C for 5 mins and incubate on ice for at least 1 min.
2. Collect all components by a brief centrifugation and add 1 µl of the EasyScript RTase to the tube.
3. Mix well and collect all the components by a brief centrifugation.
4. Incubate the tube at room temperature for 10 min for annealing.
5. Perform cDNA synthesis by incubating the tube for 50 min at 42°C.
6. Stop the reaction by heating it at 85°C for 5 min.
7. Chill on ice. The newly synthesized first-strand cDNA is ready for immediate downstream applications. 

EasyScript cDNA Synthesis Kit

Application:

• First strand cDNA synthesis for PCR
• Construction of cDNA libraries
• Generation of probes for hybridization

EasyScript cDNA Synthesis Kit is a complete system for the efficient synthesis of first strand cDNA from RNA templates. The recombinant RNasin Ribonuclease Inhibitor, supplied with the kit effectively protects RNA template from degradation.

The kit is also supplied with both oligo(dT) and random primers. The oligo(dT) anneals selectively on the poly(A) tail of mRNA. Random primers do not require the presence of poly(A). Therefore, they can be used for transcription of the 5'-end regions of mRNA. Gene-specific primers may also be used with the kit. The first strand of cDNA can be directly used as a template in PCR.

EasyScript Reverse Transcriptase (RTase) within the kit is a genetically modified form of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV). Both the EasyScript and EasyScript Plus are RNase H deficient (negative). The enzyme is purified from bacteria host as a single holoenzyme of 71 kDa with the capacity of first-strand cDNA synthesis of template up to 9kb and can be used for routine cDNA synthesis.

Kit Components:

Storage Buffer:
50mM Tris-HCl (pH 8.3), 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100, and 50% (v/v) glycerol.

Storage: 
Store at -20°C in a frost-free freezer. Multiple freezing and thawing of RNA should be avoided. Keep RNA on ice all the time. It is recommended that the first strand cDNA synthesis is carried out under conditions where RNase contamination has been eliminated.

General Protocol:

RT-PCR reactions should be assembled in a RNA-free environment. The use of "clean", automatic pipettes designated for PCR and aerosol resistant barrier tips are recommended. 
1. Thaw template RNA and all reagents on ice. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes.
2. Prepare the following reaction mixture in a PCR tube on ice:

3. Incubate at 25°C for 10 minutes if random primer is used. Omit this step if Oligo(dT) primer or sequence specific primer are used.
4. Incubate the mixture at 42°C for 60 minutes.
5. Stop the reaction by heating at 85°C for 5 minutes.
6. Chill on ice. The newly synthesized first-strand cDNA now can be used directly for PCR amplification.

Notes:
1. Isolation of poly(A)+ RNA from total RNA is not mandatory; however, doing so may improve the yield and purity of the final product.
2. RNA sample must be free of contaminating genomic DNA.
3. Unlike the oligo(dT) priming, which usually requires no optimization, the ratio of a random primer to RNA is critical in terms of the average length of cDNA synthesized in the reaction. Increasing the ratio of random primer/RNA will result in higher yield of shorter (~500 bp) cDNA, whereas decreasing this ratio will produce longer products.

4. The synthesized cDNA should be stored at -20°C.

EasyScript Reverse Transcriptase

Application:

• Synthesis cDNA froma single-stranded RNA or DNA primer extension
• Sequencing dsDNA
• cDNA library
• Template production for use in PCR
• 3'-end labeling of duplex DNA via end-filling reactions

EasyScript Reverse Transcriptase is a genetically modified form of Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV). Both the EasyScript and EasyScript Plus are RNase H deficient (negative). The enzyme is purified from bacteria h o s t as a single holoenzyme of 71 kDa with the capacity of first-strand cDNA synthesis of template up to 9kb and can be used for routine cDNA synthesis.

Kit Components:

Storage Buffer:
50mM Tris-HCl (pH 8.3), 100 mM NaCl, 0.1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100, and 50% (v/v) glycerol.

Storage: 
Store at -20°C in a frost-free freezer. Multiple freezing and thawing of RNA should be avoided. Keep RNA on ice all the time. It is recommended that the first strand cDNA synthesis is carried out under conditions where RNase contamination has been eliminated.

General Protocol:

RT-PCR reactions should be assembled in a RNA-free environment. The use of "clean", automatic pipettes designated for PCR and aerosol resistant barrier tips are recommended.

1. Thaw template RNA and all reagents on ice. Mix each solution by vortexing, and centrifuge briefly to collect residual liquid from the sides of the tubes. 
2. Prepare the following reaction mixture in a PCR tube on ice:

3. Incubate at 25°C for 10 minutes if random primer is used. Omit this step if Oligo(dT) primer or sequence specific primer are used.
4. Incubate the mixture at 42°C for 60 minutes.
5. Stop the reaction by heating at 85°C for 5 minutes. 
6. Chill on ice. The newly synthesized first-strand cDNA now can be used directly for PCR amplification.

Notes:
1. Isolation of poly(A)+ RNA from total RNA is not mandatory; however, doing so may improve the yield and purity of the final product. 
2. RNA sample must be free of contaminating genomic DNA.
3. Unlike the oligo(dT) priming, which usually requires no optimization, the ratio of a randomprimer to RNA is critical in terms of the average length of cDNA synthesized in the reaction. Increasing the ratio of random primer/RNA will result in higher yield of shorter (~500 bp) cDNA, whereas decreasing this ratio will produce longer products.

4. The synthesized cDNA should be stored at -20°C.

ENDURO VE20 Vertical Gel Electrophoresis System

Enduro VE20 Vertical Electrophoresis System (20 x 20 cm), includes glass plates, 2 x 24 well combs, cooling coil and casting base

• Large format vertical gel system
• Electroblotting and gel electrophoresis in same universal gel tank
• Quick assembly system for ease of use
• Complete system allows casting of gels and running of gels at same time increasing efficiency

The ENDURO VE20 is Labnet's new large format vertical gel electrophoresis system. It distinguishes itself in its flexibility of applications and ease of gel casting and set-up requiring only 4 screws to secure 4 gels. The same gel tank can house a large format electroblotting assembly sold separately. This system is ideal for first- and second dimension SDS-PAGE, native, preparative, gradient and high-resolution nucleic acid electrophoresis.

Innovative screw-clamp technology consists of only four screws to secure up to four 20 x 20cm gels. This vertical screw clamp technology distributes pressure evenly along the height of gel rather than in the center to eliminate plate bowing and gel compression, while maintaining a leak-proof seal during casting.

NO TANK TOP ASSEMBLY
A built-in inner buffer chamber within the PAGE insert allows set up to be completed without inclusion of an upper buffer chamber.

CASTING ADVANTAGES

• Dual purpose PAGE insert eliminates time consuming transfer of glass plates between separate casting and gel running components.
• Glass plates with bonded spacers assure clean well formation, correct alignment for leak-free casting and eliminates manually aligning spacers.
• Casting stand design ensures leak-free casting even if glass plates are misaligned.

Specifications:

ENDURO™ Semi Dry Blotter

• Western, Southern, and Northern Blots
• Rapid transfer times
• Uniform head dispersion
• Variable gel thickness
• Minimal buffer volume

The ENDURO™ Semi-Dry Blotter provides great flexibility in a lab since it can be used for all types of blotting: Western, Southern, and Northern. Blotting with the ENDURO™ Semi Dry Blotter typically can be done in 15 to 30 minutes making it a fast and easy way to do all your blotting applications. Set up procedures are easy and economical with little buffer needed. The screw down lid adjusts to varying gel thicknesses and sizes while the platinum coated electrodes provide uniform pressure ensuring even transfers. To provide your lab with total blotting solutions use with our ENDURO™ 250V, 3000mA high current power supply.

Specifications

Semi-Dry Blotting Systems, 10cm x 10cm

PR1MA™ Reverse Transcriptase, RNase H -

PR1MA™ reverse transcriptase (RT) is an RNA-dependent DNA polymerase ideal for use in RT-PCR and first-strand synthesis of complementary DNA (cDNA) for generation of cDNA libraries from single-stranded RNA, DNA, or RNA:DNA hybrids.

• Decreased RNase H activity enables longer cDNA synthesis (>5 kb).
• Lacks 3’ to 5’ exonuclease activity
• Optimal temperature at 42°C
• Temperature range: 40 to 50°C
• Heat inactivation: 70°C for 20 minutes
• Storage temperature: -20°C
• 10X reaction buffer included 

Buffer composition

• 50% glycerol
• 50 mM Tris-HCl
• 50 mM KCl
• 1 mM DTT
• 0.1  mM EDTA
• pH = 7.5

*These products are intended for research use only, not for therapeutic or diagnostic purposes in humans or animals. The safety and efficacy of these products in diagnostic or other clinical uses has not been established.