Product Number: P1331

Storage

Poly A RNA carrier is temperature stable, can ship at room temperature. Store at -20℃, Keep away from sunlight, sealed in dry place.

Description

Poly A is a synthetic nucleic acid sequence, which increase the yield of RNA or DNA during extraction process. Ethanol precipitation is commonly used to recover DNA and RNA from liquid samples. However, ethanol precipitation cannot completely recover the nucleic acid from the sample, at least 30% of the nucleic acid will lost. If the concentration of nucleic acid in the liquid sample is very low or DNA < 200bp, only 50% DNA and RNA can be recovered by ethanol precipitation. Poly A carrier is a type of synthetic polymer. Adding Poly A carrier in ethanol precipitation can significantly improve the yield of nucleic acid precipitation, and the recovery rate of trace DNA can reach 95-98%. At the same time, short primer (< 22bp) fragments and dNTP can be selectively removed for precipitation and recovery of labeled probes, and untagged dNTP can be removed. Compared to biologically derived nucleic acid precipitation aids such as glycogen and tRNA, Poly A carrier itself is free of nucleic acid pollution, DNase and RNase activity, and does not affect digestion, connection, transcription, PCR, transformation and transfection, nor does it affect DNA electrophoresis and DNA protein interaction. Poly A carrier has become the most commonly used nucleic acid precipitant.

Package

1g, 5g, or bulk.

Working concentration

A concentration of 0.5 mg/ml is recommended.

Features

• 1. Significantly improve the yield of DNA or RNA precipitation.
• 2. The recovery of trace DNA and RNA (20pg) was 95-98%.
• 3. It does not affect the enzyme cutting, linking, transcription, PCR and other reactions.
• 4. It does not affect the interaction between electrophoresis and DNA protein.

Note

Poly A carrier will increase the optical density value of RNA or DNA. Thus, when measuring the optical density value, to eliminate the influence of Poly A carrier, a blank control sample should be made according to the same experimental process (use the same reagent and Poly A carrier, but do not contain RNA or DNA samples, and dissolve the final Poly A carrier precipitation in the same solution as the sample). The optical density values of sample and blank control were measured at 260 and 280 nm. The actual optical density of the sample can be obtained by subtracting the optical density of the blank control from that of the sample. If the quantification does not need to be very accurate, it can also be estimated.

Product Number: RI039

Shipping and Storage

Stored at -20ºC, valid for two years.

Components

Description

RNase Inhibitor, Human Placenta,I.e. Ribonuclease Prohibitor, Human Placenta is a recombinant expression of human placental RNase inhibitor in Escherichia coli, which can bind to RNase A, RNase B, RNase C, and human placental RNase in a non competitive 1:1 ratio and inhibit their activity, thereby protecting RNA from degradation by these enzymes.

RNase inhibitor and Human Placenta have extremely strong inhibitory abilities against RNase A, RNase B, RNase C, and human placental ribonuclease, with Ki values as low as about 4×10^-14M. The affinity constant between antibodies and antigens is usually only 10^–6-10^-9M. And the binding between RNase inhibitor and these RNases is very rapid, almost forming complexes with these RNases at the moment of addition to inhibit their enzymatic activity.

RNase inhibitor and Human Placenta cannot inhibit RNase I T1、T2、H、U1、U2、CL3、RNase from Aspergillus、S1 Nuclease、Taq DNA polymerase,M-MLV reverse transcriptase Enzyme activity of SP6, T7, T3 RNA polymerase.

RNase Inhibitor,Human Placenta maintains its RNAse inhibitory activity within the pH range of 5-8, with the highest inhibitory activity observed at pH 7-8. RNase inhibitor requires at least 1mM DTT in the solution to maintain its activity.

This product has His tag on the N-terminus, and if necessary, RNase inhibitor in the solution will be added after the reaction is complete, Human Placenta can be detected by corresponding His antibodies or removed by magnetic beads or nickel columns adsorption.

This product belongs to the same category as Thermo’s RiboLock RNase Inhibitor and Promega’s RNasin Ribonuclease Inhibitor, both of which are recombinant human placental ribonuclease inhibitors with similar inhibitory effects on RNase (refer to Figure 1 and Figure 2).

Figure 1. RNase inhibitor, Comparison of the inhibitory effects of Human Placenta and competitor RNase inhibitor on RNase A enzyme activity. Incubate 5µg of yeast RNA with 0 or 2ng RNase A and 8, 4, 2, 1, or 0U RNase inhibitor in a 100µl reaction system (50mM MOPS, 5mM MgCl₂, pH 6.5) at 37ºC for 15 minutes. After reaction, take 20µl of reaction solution and use 1% agarose gel for electrophoretic analysis. As shown in the figure, this product has a similar inhibitory effect on RNAse activity compared to Competitor’s product. The experimental results obtained under different experimental conditions may vary slightly during actual operation, and the effects shown in the figure are for reference only.

Figure 2. Comparison of the inhibitory effects of RNase inhibitor, Human Placenta, and competitor RNase inhibitor on RNase A enzyme activity. Incubate 2µg of Hela cell total RNA with 0 or 0.5ng RNase A and 20, 10, 5, 2.5, 1.25, 0.625 or 0U RNase inhibitor in a 20µl reaction system (50mM MOPS, 5mM MgCl₂, pH 6.5) at 37ºC for 15 minutes. Immediately after reaction, all samples were electrophoretically analyzed with 1% agarose gel. As shown in the figure, this product has a similar inhibitory effect on RNAse activity compared to Competitor’s product. The experimental results obtained under different experimental conditions may vary slightly during actual operation, and the results shown in the figure are for reference only.

Source

Expressed by Escherichia coli, the source of the expressed gene is the gene encoding the enzyme in human placenta.

Application

Used for protecting RNA from degradation during processes such as cDNA synthesis, in vitro transcription, in vitro translation, and separation and purification of mRNA protein complexes; It can also be used for the identification of specific RNase activity, etc.

Unit definition

The amount of enzyme that can inhibit 50% of the activity of 5ng RNase A is defined as one activity unit.

Activity detection conditions:

100mM Tris-HCl (pH7.5), 1.2mM EDTA, 0.1mg/ml BSA, 100ng/ml RNase A, 0.1mg/ml E.coli [3H]-RNA, 50mg/ml yeast RNA, 8mM DTT。

Purity

Does not contain DNA endonucleases and exonucleases, and does not contain RNAses.

Storage buffer

20mM HEPES-KOH (pH7.5), 50 mM KCl, 5 mM DTT, 50% (v/v) glycerol。

Inactivation or inhibition

Heating at 75ºC for 10 minutes can result in complete deactivation. Heating at 70ºC for 10 minutes will still result in trace amounts of residual activity. Reagents such as SDS and urea that cause protein denaturation, as well as oxidants such as p-chloromercuribenzoate and potassium dichromate, can inhibit the binding of RNase inhibitor to RNAse.

Product Number: RT04

Storage condition

-20°C

 Component

Description

MMLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. MMLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase. The difference between this to the general MMLV RT is that the capacity to endure the heat is enhanced. It can remain the 100% activity at 50°C, it can also keep more than 80% activity even at 60°C.

Features

1. Lack RNase H activity: Weak RNase H activity High cDNA yield, can get more full-length cDNA.
2. Thermal stable: the optimum reaction temperature is 50°C, the highest is 60°C. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.
3. Wide temperature range: can reverse transcript from 37-60°C, with more than 80% of the highest activity at 42°C-60°C. customer can choose the reaction temperature freely.
4. Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.

Application

The first-strand cDNA synthesis; RT-PCR.

Source

Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine.

Unit definition

         Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37 °C is defined as one active unit (U).

Storage Buffer

         20 mM Tris-HCl (pH7.5), 200 mM NaCl, 0.25 mM EDTA, 0.01% NP-40(v/v), 2.5 mM DTT, 50% glycerol (v/v).

5×MMLV Reaction Buffer:

         [5×MMLV Reaction Buffer] 250mM Tris-HCl (pH 8.3), 15mM MgCl2, 375 mM KCl, 50mM DTT.

Product Number: RT03

Storage condition

-20°C

Component

Description

MMLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. MMLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase. The difference between this to the general MMLV RT is that the capacity to endure the heat is enhanced. It can remain the 100% activity at 50°C, it can also keep more than 80% activity even at 55°C.

Features

• Lack RNase H activity: Weak RNase H activity High cDNA yield, can get more full-length cDNA.
• Thermal stable: the optimum reaction temperature is 50°C, the highest is 55°C. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.
• Wide temperature range: can reverse transcript from 37-55°C, with more than 80% of the highest activity at 42°C-55°C. customer can choose the reaction temperature freely.
• Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.

Source

Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine.

Application

The first-strand cDNA synthesis; RT-PCR.

Unit definition

         Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37°C is defined as one active unit (U).

Storage Buffer

         20 mM Tris-HCl (pH7.5), 200 mM NaCl, 0.25 mM EDTA, 0.01% NP-40(v/v), 2.5 mM DTT, 50% glycerol (v/v).

5×Reaction Buffer:

         [5×RT Buffer] 250mM Tris-HCl (pH 8.3), 15mM MgCl₂, 375 mM KCl, 50mM DTT.

Product Number: RT01

Storage condition

-20°C

Component

Description

         MMLV Reverse Transcriptase isolated from E. coli carrying rat leukemia virus pol gene consists of a single peptide, molecule weight 71kd. MMLV Reverse Transcriptase, RNase H Minus is an RNA-dependent DNA polymerase with no detectable RNase H activity. A point mutation in the RNase H domain increases the thermostability of the enzyme and support greater cDNA yield of full-length transcripts than wild type M-MLV Reverse Transcriptase.

Features

•          Weak RNase H activity
•          High cDNA yield

Application

Synthesis of the first chain cDNA Library construction one-step RT-PCR primer extension 3′ and 5′ RACE.

Source

Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine.

Unit definition

         Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37°C is defined as one active unit (U).

Quality control

1. Absence of Endonuclease
   • 1μg of Lambda DNA is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 16 hour at 37°C. Following incubation, Lambda DNA is visualized as intact on an ethidium bromide-stained agarose gel to verify the absence of visible Endonuclease.
2. Absence of Nickase
   • 1μg of Type I supercoiled pBR322 is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 16 hour at 37°C. Following incubation, the supercoiled DNA is visualized on an ethidium bromide-stained agarose gel to verify the absence of visible nicking or cutting.
3. Absence of Exonuclease
   • 1μg of Lambda DNA / Hind III Markers is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 16 hour at 37°C. Following incubation, Lambda DNA / Hind III Markers is separated by 1% agarose gel and stained with ethidium bromide. Markers remain as intact bands without smearing.
4. Absence of RNase
   • 1ug of RNA is incubated with 200 units of MMLV Reverse Transcriptase, RNase H Minus, 1×RT Reaction Buffer for 4 hour at 37°C. Following incubation, the RNA is visualized as intact band on an ethidium bromide-stained agarose gel to verify the absence of visible RNase.
5. Function Assay
   • First-Strand cDNA Synthesis 200 units of enzyme incubated with 2.5ug of 8.3kb RNA for 1 hours at 42°C must synthesize ≥65% cDNA. ≥45% of the cDNA must be >6kb when analyzed by agarose gel electrophoresis.
6. Physical Purity
   • The purity is ≥95% as judged by SDS-polyacrylamide gel with Coomassie blue staining.

Product Number: IIA16

Storage condition

-20°C

Product component

Description

         HiFi II MMLV (H-) is a reverse transcriptase that uses engineered Escherichia coli to recombine and express mutated MMLV gene. This enzyme can catalyze complementary DNA polymerization using RNA or DNA: RNA hybrid chain as template. The mutated HiFi II MMLV (H-) reverse transcriptase RNase H activity is missing, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi II MMLV (H-) reverse transcriptase can synthesize the first strand of cDNA at 55 °C, providing higher specificity and stability. It can synthesize up to 12 kb of cDNA with high cDNA yield. Suitable for the synthesis of first strand cDNA, RT PCR, RT qPCR, and the construction of full-length cDNA libraries. The enzyme has been optimized with a unique A16 modification. IIA16 is a customized MMLV Reverse Transcriptase.

Unit definition

         Using Poly (A) as the template and oligo (dT) as the primer, the enzyme required to catalyze the incorporation of 1 nmol of dTTP within 10 minutes at 37 °C is defined as one active unit (U).

Quality control

200 U of this enzyme reacted with 1 µg of 16S, 23S rRNA at 37°C for 1 hour, and the electrophoretic bands of the RNA did not change

Note

• 1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in a dedicated area, using specialized instruments and consumables. Operators should wear masks and disposable gloves, and frequently change gloves.
• 2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37℃ for 12 hours and sterilized under high pressure at 120℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and subjected to high-pressure sterilization.
• 3. Before use, all reagents in this reagent kit should be gently mixed upside down to avoid foaming and used after brief centrifugation. The enzymes involved should be returned to -20℃ as soon as possible after use to avoid repeated freezing and thawing.
• 4. If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors.

Product Number: PK01                           Lyophilized powder     Molecular bio grade

Shipping and Storage

Shipping under room temperature and Store at 4 to -20°C recommended.

Description

Proteinase K is a broad-spectrum serine protease originally isolated from fungus Engyodontium album. Proteinase K is commonly used in molecular biological and biopharmaceutical applications to remove protein contamination from preparations of highly native undamaged nucleic acid because it rapidly and effectively inactive nuclease that might degrade the DNA or RNA even in the presence of denaturing reagents. MEBEP recombinant proteinase K is a mutant to the native protease that result in improved specific activity, higher yield and wider range of pH/temperature with optimal activity. The large scale recombinant preparation has advantages in lot-to-lot consistency, superior purity and cost-efficiency. DNA-free nature of recombinant Proteinase K made it well-suited in isolating DNA and RNA templates. Recombinant proteinase K is widely used for general digestion of proteins and Chemo-enzymatic peptide synthesis in molecular biology, molecular diagnostic and biopharmaceutical applications. Proteinase K is one of important enzymes in various industries like leather, cosmetics, food and feed etc.

Preparation

1. The Molecular Biology Grade lyophilized powder is NOT sterile.
2. Stock solution can be prepared as 40-80 mg/ml in dilution buffer [20mM Tris-HCl (pH 7.4) , 1 mM CaCl₂] or [20mM Tris-HCl (pH 7.4) , 1 mM CaCl₂, 2% Glycerol], sterilized using a 0.22 μm filter and supplied at final concentration of 20-40mg/ml in 50% sterilized Glycerol. Store in aliquots at wide temperature range from 24°C to -80°C.
3. PES or PVDF membranes with low protein binding are recommended in sterile filtration.

Quality Control

1. DNase Activity: none detectable enzyme activity with λ DNA after 6 hours incubation at 37°C.
2. RNase Activity: none detectable of ribonuclease activity after 16 hours incubation at 25°C.
3. Protein Purity: > 95% (Native-PAGE and SDS-PAGE assay).

Product Number: UG01

Shipping and Storage

-20°C.

Components

Description

Uracil-N-glycosylase (UNGase), also known as uracil DNA glycosylase (UDGase), is a recombinant enzyme expressed and purified by E. coli. The protein has a molecular weight of 25 kD and can catalyze uracil-containing Single- and double-stranded DNA releases free uracil and is inactive to RNA, and is mainly used to prevent contamination of PCR amplification products. The mechanism of action of the enzyme is as follows: in the PCR reaction, dUTP is used instead of dTTP, and all Ts in the amplified product fragments are replaced by U, forming a PCR amplification product containing dU bases. UNG enzyme can selectively break the glycosidic bond of U base in single-stranded and double-stranded DNA, degrade the DNA containing U in the reaction system, effectively eliminate the residual contamination of PCR products, and greatly reduce false positives caused by contamination of amplification products, thereby ensuring specificity and accuracy of amplification.

Activity Definition

One unit is defined as the amount of enzyme required to catalyze 1nmol of uracil from uracil-containing DNA in 60 minutes at 37°C.

Quality Control

The purity detected by SDS-PAGE was more than 95%; no endonuclease or exonuclease activity was detected

Description

MMLV Reverse Transcriptase, encoded by Moloney Murine Leukemia Virus (MMLV RT) is an RNA-dependent DNA polymerase that synthesizes the complementary cDNA first strand from a single-stranded RNA template to which a primer has been hybridized. MMLV RT will also extend primers hybridized to single-stranded DNA. Second strand cDNA synthesis can be achieved from some mRNA templates without an additional DNA polymerase. The difference between this to the general M-MLV RT is that the capacity to endure the heat is enhanced. It can remain the 100% activity at 50°C, it can also keep more than 80% activity even at 55°C.

Source

Recombination of E. coli containing Moloney murine leukemia virus reverse transcriptase gene from clone of Moloney murine.

Applications

The first-strand cDNA synthesis; RT-PCR

Features

• lack RNase H activity: Weak RNase H activity High cDNA yield, can get more full length cDNA.
• thermoStable: the optimum reaction temperature is 50°C, the highest is 60°C. Can overcome the template RNA secondary structure, and finish the reverse transcriptase experiment smoothly.
• wide temperature range: can reverse transcript from 37-60C, with more than 80% of the highest activity at 42°C-55°C customer can choose the reaction temperature freely.
• Strong amplification activity: Gene mutation enhanced the binding capacity of the enzyme and RNA. Increased the amplification speed, can obtain the quality cDNA, suitable for cDNA library construction.

Unit Definition

One unit of MMLV RT catalyzes the incorporation of 1 nmol of dTTP into acid insoluble material in 10 minutes at 37°C using oligo(dT)12-18-primed poly(A)n as a template.

Concentration: 200U/μl

Package: Bulk

Component: M-MLV (200U/μl) 5xBuffer (with DTT)

Storage: -20°C

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Description

The Hot Start Taq DNA Polymerase is a recombinant Taq DNA polymerase which has been chemically modified by the addition of heat-labile blocking groups to its amino acid residues. The enzyme is inactive at room temperature, avoiding extension of non-specifically annealed primers or primer dimers and providing higher specificity of DNA amplification. The functional activity of the enzyme need to be activated in incubation at 95°C for 10 minute. The activated enzyme maintains the same functionality.

Taq DNA polymerase: Has the 5′ to 3′ DNA polymerase activity and 5′ to 3′ exonuclease activity without 3′ – 5′ exonuclease activity. The extending speed is 1 kb/30 sec. There is an “A” on 3′ end. The PCR product can be cloned in TA vector.

Applications

• Hot-start PCR amplification
• Specific amplification of complex cDNA and genomic template
• Amplification from low copy number DNA template
• Real-Time PCR
• Multiple PCR
• Generation of PCR products for TA cloning, Gene microarray analysis and SNP test

Quality Control

• Functional absence of double and single-stranded endonuclease activity;
• Purity>99% test by SDS gel electrophoresis;
• Each lot of HotStar Taq DNA Polymerase is assayed for amplification from as little as 10 ng of human genomic DNA;
• Retain full activity at room temperature for one week;
• No host DNA residue.

Concentration: 5 u/μl

Package: Bulk

Storage: Store at -20°C

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