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PR1MA One-Step RT-PCR Kit
  • Convenient cDNA synthesis and PCR in a single tube from 1 pg total RNA
  • Formulated for highly specific and sensitive RT-PCR from any RNA templates
  • Incorporates thermostable reverse transcriptase and Hot-Start Taq for preparation at room temperature


The PR1MA One-Step RT-PCR Kit has been formulated for cDNA synthesis and subsequent PCR in a single tube for end-point analysis. This latest generation RT-PCR Kit consists of an MMLV-derived, thermostable Reverse Transcriptase (45°C to 55°C), an advanced RNase Inhibitor and PR1MA Hot Start Taq for ultra-sensitive one-step RT-PCR from as little as 1pg total RNA starting material.

The optimized buffer chemistry allows for efficient reverse transcription and PCR of problematic sequences with significant secondary structure (such as GC-rich targets). The PR1MA One-Step RT-PCR Kit is ideal for determining the presence or absence of RNA templates and quantifying expression through qualitative analysis of RNA transcription levels. The kit also efficiently synthesizes double-stranded cDNA for subsequent gene expression analysis.

PR1MA™ qMAX™ cDNA Synthesis Kits

PR1MA™ now offers two cDNA Synthesis Kits, to meet a range of requirements.
 

The original cDNA Synthesis Kit is a 2-tube format for easy reaction setup and is ideal for 4 pg to 0.5 µg of input RNA. One tube includes our exceptionally stable Reverse Transcriptase combined with a potent RNAse inhibitor, and the other tube contains a 5X reaction buffer with an optimal mixture of anchored oligo (dT) primers and random hexamers to produce a non-biased population of cDNA. 
 

The 1st Strand cDNA Synthesis Flex Kit includes four components: a high-capacity Reverse Transcriptase, an optimized 5X buffer, separate solutions of oligo (dT) primers, and random hexamer primers. This multi-component format is ideal for 10pg to 2.0µg of input RNA and allows for greater flexibility in assay design.

 
PR1MA™ qMax cDNA Synthesis Kits
 
 
ItemDescriptionVolume (20 µL)
PR2100-C-SPR1MA™ qMax™ cDNA Synthesis Kit10 Reactions (Sample)
PR2100-C-25PR1MA™ qMax™ cDNA Synthesis Kit25 Reactions
PR2100-C-100PR1MA™ qMax™ cDNA Synthesis Kit100 Reactions
PR2100-C-250PR1MA™ qMax™ cDNA Synthesis Kit250 Reactions
 

PR1MA™ qMax First Strand cDNA Synthesis Flex Kits  
 
ItemDescriptionVolume (20 µL)
PR2110-SPR1MA™ qMax™ First Strand cDNA Synthesis Flex Kit10 Reactions (Sample)
PR2110-50PR1MA™ qMax™ First Strand cDNA Synthesis Flex Kit50 Reactions
PR2110-100PR1MA™ qMax™ First Strand cDNA Synthesis Flex Kit100 Reactions
PR2110-200PR1MA™ qMax™ First Strand cDNA Synthesis Flex Kit200 Reactions


 Item PR2110-100 has been discontinued by the manufacturer;
however, we will be continuing to supply the other quantities.

 

In place of item PR2110-100, please order items PR2110-50 or PR2110-200

 

PR1MA™ Reverse Transcriptase

 

PR1MA™ reverse transcriptase (RT) is an RNA-dependent DNA polymerase that can be used for complementary DNA (cDNA) synthesis from an RNA template and is ideal for use in molecular amplification assays. PR1MA™ RT is a robust enzyme that works in a broad range of temperatures (40 - 72 °C) and has RNase H activity.

  • Optimal temperature: 55 °C
  • Heat inactivation: 75 °C for 20 minutes
  • Glycerol-free buffer available
  • Storage temperature: -20 °C (standard buffer)
  • 10X Isothermal buffer included

Standard Buffer Composition

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

Important note: Please be sure to use the buffer provided with this product to ensure optimal results.

 

*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.   
 

RR1MA™ CRISPR/Cas9 Nucleases

 

The RNA-guided endonuclease Cas9, associated with Type II CRISPR/Cas systems, site-specifically digests DNA using a single guide RNA (sgRNA) which it binds to direct it to the complementary sequence. MIDSCI™ offers two traditional versions of Streptococcus pyogenes Cas9 nuclease: CRISPR/Cas9, ideal for in vitro DNA digestion, and CRISPR/Cas9 NLS, for in vivo nuclear localization.

  • Optimal temperature: 37 °C
  • Heat inactivation: 65 °C for 20 minutes
  • Storage temperature: -20 °C

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.

 

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.

Target DNA

x µl (100ng)

sgRNA

x µl (4000ng)

10x Cas9 Reaction Buffer

3.0 µl

Cas9 Nuclease

1.0 µl (160ng)

Add H2O up to

30.0 µl

Item#:
ASCDNART6

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.

  


Component

10 µl Reaction

Vector DNA

x µl

Insert DNA

x µl

10 mM ATP

1.0µl

10x T4 Ligase Buffer

1.0µl

T4 DNA Ligase

1.0µl

Add H2O up to

10.0µl

  

  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.
Item#:
ASCDNART8

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.  


Linearized vector

x µl (50-100 ng)

Insert

x µl (50-100 ng)

5x ig-Fusion enzyme premix

2.0 µl

H2O up to

10.0 µl

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).

Item#:
ASCDNART7

Need a great Reverse Transcriptase product at a great price? Try PR1MA!  
Click here for PCR or here for qPCR to order.


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:

EasyScript R Tase (200U/µL): 100L
2X Reaction Mix: 1200µL
Nuclease-Free H2O: 2 x 1mL


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

 


Order#

Description

Quantity

Rxn

BERTCDNA-25

RT 2X Master Mix  

250µl

25 rxns

BERTCDNA-100

RT 2X Master Mix 

1ml

100 rxns

  • 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:

    Components

    Volume

    Final Conc.

    Total RNA, or

    Variable

    1 ng - 2 µg/rxn

    mRNA

    Variable

    1 pg - 2 ng/rxn

    2X Reaction Mix

    10 µl

    1X

    H2O

    Up to 19 µl

    -

    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.
Item#:
ASCDNART1

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EasyScript cDNA Synthesis Kit

Order#

Description

Quantity

Rxn

G233

EasyScript cDNA Synthesis Kit

5000U (25uL)

25 rxns

G234

EasyScript cDNA Synthesis Kit

20,000U (100uL)

100 rxns

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:

Components

EasyScript cDNA Synthesis Kit

 

G233

G234

EasyScriptRTase (200U/µl)

5,000U

20,000U

Oligo(dT) (10µM)

40 µl

160 µl

Random Primers (10µM)

40 µl

160 µl

5x RT buffer

150 µl

600 µl

RNasin (40U/µl)

15 µl

60 µl

dNTP (10mM)

40 µl

160 µl

RNase-free H2O

1 ml

2x1 ml

Size

25 rxns

100 rxns

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:

 

Volume

Concentration (final 20µl)

Total RNA, or poly(A)+RNA

Variable

0.5-5µg per reaction

 

 

50ng-0.5µg per reaction

Oligo(dT) (10µM)

1µl

0.5µM

or Random Primer (10µM)

1µl

0.5µM

or Sequence-specific Primer

Variable

10-15pM

dNTP (10mM)

1µl

500µM

5X RT Buffer

4µl

1X

RNasin (40U/µl)

0.5µl

20U per reaction

EasyScript RTase (200U/µl)

1µl

200U per reaction

RNase-free H2O

Variable

-

Final volume

20µl

-

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.

Item#:
ASCDNART4

Need a great Reverse Transcriptase product at a great price? Try PR1MA!  
Click here for PCR or here for qPCR to order.


EasyScript Reverse Transcriptase

Order#

Description

Quantity

Rxn

G231

EasyScript Reverse Transcriptase

5000U (25uL)

25 rxns

G232

EasyScript Reverse Transcriptase

20,000U (100uL)

100 rxns

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:

Components

EasyScript Reverse Transcriptase

 

G231

G232

EasyScript RTase (200U/µl)

5,000U

20,000U

5x RT buffer

150 µl

600 µl

Size

25 rxns

100 rxns


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:

 

Volume

Concentration (final 20µl)

Total RNA, or poly(A)+RNA

Variable

0.5-5µg per reaction

 

 

50ng-0.5µg per reaction

Oligo(dT) (10µM)

1µl

0.5µM

or Random Primer (10µM)

1µl

0.5µM

or Sequence-specific Primer

Variable

10-15pM

dNTP (10mM)

1µl

500µM

5X RT Buffer

4µl

1X

RNasin (40U/µl)

0.5µl

20U per reaction

EasyScript RTase (200U/µl)

1µl

200U per reaction

RNase-free H2O

Variable

-

Final volume

20µl

-

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.

Item#:
ASCDNART3

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.

 

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