Bullseye 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
 
50 mM 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.

Isohelix™ Proteinase K

• Available in Different Quantities
• Light Reducing Container
• Suitable for -20ºC Storage
• High Quality – Fully QC Tested

Lyophilized proteinase K is ideal for the Genefix™ Saliva Kits. Comes with instructions.

 

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

EDTA Disodium Salt

• Appearance: White Crystalline Powder
• Purity: ≥ 99.0%
• pH (5% Aqueous): 4.0 - 6.0
• Solubility: Clear and Colorless to Faint Yellow
• Identification (FTIR): Conforms to Structure
• Loss on Drying: 1.0%
• Insoluble Matter: ≤ 0.005%
• Nitriloacetic Acid: ≤ 0.1%
• Lead: ≤ 5 ppm
• Iron: 10 ppm
• DNase: None Detected
• RNase: None Detected

*Custom sizes available upon request.

DOT Information: Non-regulated.   
 

• Mix of dATP, dCTP, dGTP, dTTP
• Each nucleotide is at a concentration of 100 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 

dNTP SET

(dATP, dCTP, dGTP, dTTP)

100 mM

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

Sodium Dodecyl Sulfate

• Appearance: White Powder
• Purity: ≥ 98.0%
• Active Matter: ≥ 99%
• Identity (FTIR): Conforms to Structure
• Lead: ≤ 5 ppm
• Phosphate: ≤ 1ppm
• A260 (3% Aqueous): ≤ 0.4
• A280 (3% Aqueous): ≤ 0.1
• RNase: None Detected
• DNase: None Detected

*Custom sizes available upon request.

DOT Information: Non-regulated.   
 

Pfu 2x Master Mix 
Pfu DNA Polymerase 2x master mix is ready to use premix which contains Pfu DNA Polymerase, dNTPs, MgCl2 and stabilizers with optimized reaction buffer. It has been optimized for routine PCR applications. Pfu DNA Polymerase is a heat stable DNA polymerase which has 5' 3' DNA polymerase and 3' 5' exonuclease (proofreading) activities. Pfu DNA polymerase retains the high fidelity, sensitivity and processivity with an error rate six-fold lower than Taq DNA polymerase, and significantly lower than the error rates of most other proofreading enzymes or DNA polymerase mixtures. Pfu 2x Master Mix product is supplied with the unique Intact Genomics 5x Magic Enhancer that enables efficient amplification of GC rich templates up to 84%.

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. 

Product Source
E. coli strain expressing a Pfu DNA Polymerase gene from Pyrococcus furiosus.

Contents & Storage

1. Pfu 2x master mix
2. 5x Magic Enhancer

Store all contents at -20 °C.

1x Master Mix Composition 
10 mM Tris-HCl pH 9.0, 50 mM KCl, 1.5 mM MgCl, 0.2 mM dNTPs, 5% Glycerol, 0.08% Igepal CA 630, 0.05% Tween-20, 100 Units/ml Pfu DNA Polymerase.

Protocol
1. Prepare a reaction mix according to the following table:
  

2. Mix the reaction mixture thoroughly.
3. Program the thermal cycler according to the manufacturer's instructions.
4. A typical PCR cycling program is outlined in the following table.

5. Place the PCR tubes in the thermal cycler and start the cycling program.

6. Analyze 5 µl of PCR products by agarose gel electrophoresis. 

Pfu DNA Polymerase 
Pfu DNA polymerase is a heat stable DNA polymerase which has 5' 3' DNA polymerase and 3' 5' exonuclease (proofreading) activities. Pfu DNA polymerase retains the high fidelity, sensitivity and processivity with an error rate six-fold lower than Taq DNA polymerase, and significantly lower than the error rates of most other proofreading enzymes or DNA polymerase mixtures (1). This product is supplied with the unique Intact Genomics 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM, and 5X Magic Enhancer that enables efficient amplification of GC rich templates up to 84%. The physical purity of this enzyme is 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. 

Product Source
E. coli strain expressing a Pfu DNA Polymerase gene from Pyrococcus furiosus.

Contents & Storage

1. Pfu DNA Polymerase
2. 10x PCR Buffer with Mg²+
3. 5x Magic Enhancer
4. 10 mM dNTP (Cat. # 3312d, 3314d only)

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 PCR Buffer with Mg2+ 
100 mM Tris-HCl pH 9.0, 15 mM MgCl, 100 mM KCl, 80 mM (NH4)2SO4, 0.5% Igepal CA 630

Unit Definition 
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTP into acid-insoluble form in 30 minutes at 72 °C.

Protocol
1. Thaw 10x PCR Buffer, dNTP mix, primer solutions, 5x Magic Enhancer (if required) and mix thoroughly before use. 
2. Prepare a reaction mix according to the following table: The reaction mix typically contains all the components needed for PCR except the template DNA.

3. Mix the reaction mixture thoroughly.
4. Add template DNA to the individual PCR tubes containing the reaction mixture. 
5. Program the thermal cycler according to the manufacturerâs instructions. A typical PCR cycling program is outlined in the following table.

This product is no longer available, please contact us for other options.
 

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Visit our PCR Reagents page for more options! 

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

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.

2) Gently mix the reaction mixture and centrifuge briefly.
3) Incubate at 37 °C for 60 min. 
4) Add 1 µl RNase (4 mg/ml) 
5) Incubate at 37 °C for 20 min.
6) Run 0.7 to1% agarose TBE gel.

Bullseye PREMIUM HS-Taq DNA Polymerase Master Mix Blue

HS Taq Polymerase Master Mix BLUE is a ready to use 2 X master mix. Simply add primers, template and water to successfully carry out primer extensions. The master mix is available in 100, 500, 1000, and 2500 reaction sizes and is available with two different buffer options (HS Buffer 1 and HS Buffer 2)

HS Taq Polymerase is a modified form of Bullseye Taq, activated by heat treatment. This results in higher specificity and greater yields when compared to standard Taq. The added benefit of the HS Master Mix Blue is that it can be loaded directly onto the gel as there is no need to use separate loading dyes for subsequent electrophoresis and visualization. This cuts down on the chance of contaminating the component stocks and leads to better reproducibility.

Composition of 2 X HS Master Mix Blue with Buffer 1
Tris-HCl, pH 8.5, (NH4)2SO4,3 mM, MgCl2, .2 % Tween 20, .4 mM dNTPs, .2 units/uL HS Taq Polymerase, Inert Blue Dye, Stabilizer

Composition of 2 X HS Master Mix Blue with Buffer 2
Tris-Hcl, pH 8.5, Balanced KCl/(NH4)2SO4,3 mM, MgCl2, .2 % Tween 20, .4 mM dNTPD, .2 units/uL HS Taq Polymerase, Inert Blue Dye, Stabilizer

Bullseye Taq DNA Polymerase

Bullseye Taq DNA Polymerase is the most popular thermostable enzyme used in DNA amplification experiments. This high performance Taq DNA polymerase is specifically purified to produce excellent yields with little or no background. Its outstanding activity and unique thermal DNA amplification properties make it one of the best-value DNA polymerases available.

• Robust performance
• Leaves 3' A overhang
• Stable at all storage temperatures
• Ideal for general PCR, genomic analysis and TA cloning

Storage: -20°C

Quality Control
Taq DNA Polymerase is highly purified, free of contaminating endonucleases, exonucleases and nicking activity. For endonuclease assay, 1 ug of Lamda/Hind III DNA is incubated with 20 units of enzyme in assay buffer at 75oC for 16 hours with no visible contaminating activity observed. Also, every lot is tested for its performance consistency.

Unit Definition
One unit incorporates 10n moles of 4 radioactive labeled dNTPs into acid-insoluble material in 30 minutes at 74°C.

Storage Buffer
5 units / uL in 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol, 0.5% TritonX-100, and 0.5% NP-40.
10x Reaction Buffer 100 mM KCl, 100 mM Tris HCl (pH9.0), 80 mM (NH4)2SO4, and 1.0% Triton X-100. (Mg++free): is optimized for use with 200 uM dNTPs.

Magnesium Chloride
25 mM MgCl2: In general, 1.5 mM MgCl2 is recommended; this may vary with different conditions and primer sets.
 
*For laboratory research only. Not for clinical applications. 

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