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Thursday, May 15, 2014

Product Focus: Taq DNA Polymerase

Taq DNA Polymerase is a thermostable enzyme which has a highly processive 5′→3′ polymerase activity and a 5′→3′ exonuclease activity. Taq is functionally tested and has no detectable contaminating endonucleases or exonucleases. Taq DNA Polymerase withstands repeated incubations at 95°C without a significant decrease in enzyme activity, and is suitable for routine PCR. Taq DNA Polymerase is supplied with 10X PCR Reaction Buffer plus a separate tube of 25mM MgCl2 for optimizing PCR conditions. Each lot is tested for product yield and length in PCR amplification.

View Hi-Res PDF Version
Figure 1. DNA fragments ranging from 1 kb to 6 kb amplified from λ DNA.

Properties:  Activator: Mg2+

Purity: 
Free from detectable non-specific nucleases.
Storage Buffer:  20mM Tris-HCl, pH 8.5, 1mM DTT, 0.1mM EDTA, 100mM KCI, 50% glycerol, stabilizers.
Unit Definition:  One unit incorporates 10 nmol of total nucleotides into acid-insoluble material in 30 min at
74°C in a total volume of 50 μl.
Concentration:  5 units/μl
Assay Conditions:  The reaction mixture (50 μl) contains 25mM TAPS, pH 9.3 (at 25°C), 50mM KCl, 2mM MgCl2, 1mM 2-mercaptoethanol, 200μM dNTPs, 250 μg/ml activated salmon sperm DNA, and Taq DNA Polymerase. After incubation at 74°C for 10 min, acid insoluble material is determined.
Functional Test:  Tested in a standard PCR.
Functionally Tested 10X PCR Reaction Buffer (included, PN 71165):  100mM Tris-HCl (pH 8.6), 500mM KCl, 15mM MgCl2
Functionally Tested MgCl2 (included, PN 71167):  25mM solution
Applications:
  • Routine PCR amplification
  • Suitable for real time qPCR


References

  1. INNIS, M. A., MYAMBO, K. B., GELFAND, D. H. AND BROW, M. A. (1988) Proc. Natl. Acad. Sci. 85, 9436-9440.
  2. LAWYER, F. C., STOFFEL, S., SAIKI, R. K., MYAMBO, K., DRUMMOND, R. AND GELFAND, D. H. (1989) J. Biol. Chem. 264, 6427-6437.
  3. INNIS, M. A. AND GEFLAND, D. H. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press.

 

Technical Information


Products

Functionally tested and has no detectable contaminating endonucleases or exonucleases
Catalouge No. Description Unit Size
71160 50 UN
Taq DNA Polymerase
50 units
71160 250 UN
Taq DNA Polymerase
250 units
71160 1000 UN
Taq DNA Polymerase
1000 units
71160 5000 UN
Taq DNA Polymerase
5000 units


Where can I find more information about Affymetrix USB?
 
Visit the manufacturer page at www.stratech.co.uk/affymetrix, email info@stratech.co.uk or call +44 (0) 1638 782600.
 
Stratech Scientific is a distributor of high quality, competitively priced, reliable products for research laboratories throughout the UK and Europe. Please contact us to find out which ranges we can supply in your country.


Product Focus: ExoSAP-IT® For PCR Product Cleanup

  • Conserves PCR samples — 100% recovery of both short and long PCR products
  • One tube/one step PCR cleanup — Add ExoSAP-IT reagent directly to PCR product
  • Eliminates spin columns — Decreases time and expense while increasing yield
  • Removes contaminating primers and dNTPs — No interference in downstream applications
  • Scalable — Economical for high-throughput purification
  • Simple processing — Robotic-friendly; Replaces beads, filtrations, and plates
  • Generates less waste than columns
ExoSAP-IT reagent is designed for simple, quick PCR cleanup for downstream applications, such as DNA sequencing or Single Nucleotide Polymorphism (SNP) analysis. When PCR amplification is complete, any unconsumed dNTPs and primers remaining in the PCR product mixture will interfere with these methods. ExoSAP-IT removes these contaminants.


Additional Information

ExoSAP-IT is added directly to the PCR product and incubated at 37°C for 15 minutes (Fig. 1). After PCR treatment, ExoSAP-IT is inactivated simply by heating to 80°C for 15 minutes.
View Hi-Res PDF Version
Fig. 1. Summary of ExoSAP-IT PCR product treatment.
ExoSAP-IT single-step PCR cleanup utilizes two hydrolytic enzymes, Exonuclease I and Shrimp Alkaline Phosphatase (SAP), together in a specially formulated buffer, to remove unwanted dNTPs and primers from PCR products. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced in the PCR. SAP removes the remaining dNTPs from the PCR mixture.

Rapid PCR Product Cleanup Protocol
ExoSAP-IT requires only one pipetting step and two incubations. Just add ExoSAP-IT to the PCR product and within 30 minutes sequencing or SNP analysis can be performed.

Simple: Single-Step
The method is designed to require a minimum of ‘hands-on’ time. Enzymatic removal of excess primers and unincorporated nucleotides occurs in one easy step by using ExoSAP-IT reagent in a single tube or microtiter well. Only simple pipette transfers are required, therefore, many samples can be processed at once, either manually or with robotics.

No Sample Loss
Use of ExoSAP-IT reagent eliminates all gel or column purifications, sedimentations, filtrations, beads, and/or magnetic separations(1). There is 100% recovery of both short and long PCR products with ExoSAP-IT (Fig. 2).

View Hi-Res PDF Version
Fig. 2. ExoSAP-IT treatment of PCR products with no sample loss. Single-copy targets were amplified from human genomic DNA. HES-1 (125 bp), numb (455 bp), NRAGE (1.55 kb), and numb (4.6 kb) were loaded on a 1.5% agarose gel before (pre) and after (post) ExoSAP-IT treatment. M is the DNA marker lane. Note that a variety of PCR product sizes can be treated with ExoSAP-IT, even a 125 bp fragment, with no sample loss. 

Achieve High Data Quality from PCR Products
ExoSAP-IT reagent may be used as an effective cleanup method prior to fluorescent or radioactive DNA sequencing, SNP analysis, or any other application requiring a PCR product free of excess nucleotides and primers.

Fig. 1. Fluorescent sequencing results of a 1,007 bp PCR product. A 1,007 bp fragment was amplified and treated with ExoSAP-IT reagent (above) or an alternate enzymatic reagent (below) and sequenced. Pherograms revealed no miscalls with ExoSAP-IT reagent but four miscalls at position 25, 26, 30, and 39 with the alternate enzymatic reagent. 5 µl of PCR product was treated with 2 µl ExoSAP-IT reagent or the alternate enzymatic method per its protocol. Note: Two of the four miscalls are frame-shifts.
Fig. 2. Fluorescent sequencing results of a 100 bp pUC18 PCR fragment sequenced with a -20 Fwd primer using fluorescent sequencing reagents. PCR cleanup performed with: (a) ExoSAP-IT reagent; (b) a column designed for PCR cleanup. Base miscalls in (b) are due to inherently low yields of short PCR products when using columns.

References:
  1. DUGAN, K. A., LAWRENCE, H. S., HARES, D. R., FISHER, C. L. AND BUDOWLE B. (2002) J. Forensic Sci 47, 811-818.
  2. HANKE, M. AND WINK, M. (1994) BioTechniques 17, 858-860.
  3. MU, J., DUAN, J., MAKOVA, K., JOY, D., HUYNH, C., BRANCH, O., LI, W. AND SU, X. (2002) Nature 418, 323-326.
  4. SILVA, JR., W. A., COSTA, M. C. R., VALENTE, V., DE FREITAS SOUSA, J., PACÓ-LARSON, M. L., ESPREAFICO, E. M., CAMARGO, S. S., MONTEIRO, E., DE JESUS, A., HOLANDA, M. A., ZAGO, M. A., SIMPSON, A. J. G. AND NETO, E. D. (2001) BioTechniques 30, 537-542.
  5. WERLE, E., SCNEIDER C., RENNER, M., VÖLKER, M. AND FIEHN, W. (1994) Nucleic Acids Res. 22, 4354-4355.
*5 µl of PCR product was treated with 2µl ExoSAP-IT reagent or 1µl Exo I and 1µl Alkaline phosphatase from an alternative enzymatic method using the recommended incubation conditions (15 minutes at 37°C followed by 15 minutes at 80°C) in 16 duplicates each. The treated PCR products were then sent for automated sequencing. Sequencing results over the first 600 bp consistently show more miscalls with the samples treated with the alternative enzymatic method than samples treated with ExoSAP-IT reagent. The pherogram shown here is an example. Product sequenced after using the alternative enzymatic method showed 3 miscalls and a 1 base frame shift. The same sequence after ExoSAP-IT reagent treatment was completely accurate with no miscalls or errors.


Technical Information



Where can I find more information about Affymetrix USB?
 
Visit the manufacturer page at www.stratech.co.uk/affymetrix, email info@stratech.co.uk or call +44 (0) 1638 782600.
Stratech Scientific is a distributor of high quality, competitively priced, reliable products for research laboratories throughout the UK and Europe. Please contact us to find out which ranges we can supply in your country.

Thursday, April 24, 2014

Product Focus: Thermophilic Polymerases for Maximum Flexibility

Jena Bioscience offers a broad range of optimized thermophilic polymerases. Make your selection from Taq polymerases for routine PCR applications, hot start polymerases for high specific amplifications or proofreading enzyme blends for high fidelity and long range PCR. All enzymes ensure reliable, high performance results and guarantee maximum success for their particular application.

Standard PCR – Product Selection Guide


 

Taq Pol – Thermostable DNA polymerase

 


 

Taq Pol / high yieldThermostable DNA polymerase

Taq Pol / high yield is recommended for use in routine PCR reactions. The buffer system is optimized for high efficiency and gives superior amplification results in a broad range of reaction conditions with most primer-template pairs. The buffer system facilitates the incorporation of labeled or modified nucleotides into DNA. Note that the ammonium based buffer contains detergents and may interfere with automated pipetting systems.
The enzyme replicates DNA at 72°C. It catalyzes the polymerization of nucleotides into duplex DNA in 5′→3′ direction in the presence of magnesium. It also possesses a 5′→3′ polymerization-dependent exo-nuclease replacement activity but lacks a 3′→5′ exonuclease activity.
Taq Pol – red / high yield buffer contains an inert red dye for easier handling and to facilitate the preparation of the master mix.


 

Hot Start PolHeat-activatable DNA polymerase for high specificity

  • Hot Start Pol provides improved specificity and sensitivity when amplifying low-copy-number targets in complex backgrounds or when prolonged room-temperature set up is required. The polymerase activity is blocked at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of nonspecifically annealed primers and primer-dimer formation at low temperatures during PCR setup.
    The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5′→3′ direction in the presence of magnesium. It also possesses a 5′→3′ polymerization-dependent exonuclease replacement activity but lacks a 3′→5′ exonuclease activity. 
  • Hot Start Pol requires no prolonged activation step. The polymerase inhibiting ligand is quickly released at the increased temperature of thermal cycling. 
  • Hot Start Pol – red contains an inert red dye for easier handling and to facilitate the preparation of the master mix.


 

Rapid Pol - Thermostable DNA polymerase for rapid PCR

Rapid Pol is based on a specifically processed enzyme engineered for higher processivity and speed, offering significantly faster extension rates than wild-type Taq polymerase.
The rapid polymerase is designed for Fast PCR, in which total reaction times are 20-70% shorter compared with conventional PCR assays. This can be achieved without sacrificing reaction performance or the requirement for specialized PCR consumables or thermocyclers.


 

High Fidelity PolDNA polymerase for high accuracy

High Fidelity Pol is based on a blend of Taq DNA polymerase and a proofreading enzyme specially designed for highly accurate and efficient amplification. It shows excellent results with extremely long (up to 30 kb), GC-rich or other difficult templates.
The enzyme blend includes a highly processive 5′→3′ DNA polymerase and possesses a 5′→3′ polymerization-dependent exonuclease replacement activity. Its inherent 3′→5′ exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase.
The enzyme is highly purified and free of bacterial DNA.


 

High Fidelity Hot Start PolHeat-activatable DNA polymerase for high accuracy and specificity

High Fidelity Hot Start Pol is based on a blend of Taq DNA polymerase and a proofreading enzyme specially designed for highly accurate and efficient amplification. The additional hot-start function provides improved specificity and sensitivity when amplifying low-copy-number targets in complex backgrounds or when prolonged room-temperature set up is required. The polymerase activity is blocked at ambient temperature and switched on automatically at the onset of the initial denaturation. The thermal activation prevents the extension of nonspecifically annealed primers and primer-dimer formation at low temperatures during PCR setup.
The enzyme shows excellent results with extremely long (up to 30 kb), GC-rich or other difficult templates.
Activation step
High Fidelity Hot Start Pol requires no prolonged heating or denaturing step. The polymerase inhibiting antibodies are released at the increased temperature of the initial denaturation.


 

Pfu-X PolymeraseProofreading DNA polymerase for highest accuracy

Pfu-X Polymerase is the ideal choice for applications where the efficient amplification of DNA with highest fidelity is required.
The enzyme is a genetically engineered Pfu DNA polymerase, but showing a 2-fold higher accuracy and an increased processivity, resulting in shorter elongation times.
The enzyme catalyzes the polymerization of nucleotides into duplex DNA in 5′→3′ direction but does not possess a 5′→3′ exonuclease replacement activity. Its inherent 3′→5′ exonuclease proofreading activity results in a greatly increased fidelity of DNA synthesis compared to Taq polymerase. Pfu-X Polymerase-generated PCR fragments are blunt-ended.
The enzyme is highly purified and free of bacterial DNA.


 

Sequencing Pol - Taq Pol mutant for incorporation of ddNTPs

Sequencing Pol is a Taq polymerase mutant showing an enhanced efficiency for incorporation of ddNTPs. Its capability of incorporating ddNTPs and dNTPs at equal rates makes it the ideal choice for DNA cycle sequencing.
The enzyme replicates DNA at 74°C. It catalyzes the polymerization of nucleotides into duplex DNA in 5′→3′ direction in the presence of magnesium and has a 5′→3′ polymerisation-dependent exonuclease replacement activity only.



Where can I find more information about Jena Bioscience?
Visit the manufacturer page at www.stratech.co.uk/jena_bioscience, email info@stratech.co.uk or call +44 (0) 1638 782600.

Stratech Scientific is a distributor of high quality, competitively priced, reliable products for research laboratories throughout the UK and Europe. Please contact us to find out which ranges we can supply in your country.