NEW PARADIGM of BIOTECHNOLOGY -GENET BIOgenetbio.co.kr/FM/protein/Brochure.pdf · 2005. 9. 30. ·...

NEW PARADIGM of B IOTECHNOLOGY

-GENET BIO

GeNet BioGloba l Gene Network

GENET BIO DNA AMPLIFICATION PRODUCTS GUIDE

Keynote of Products

Prime TaqTM DNA Polymerase

Prime TaqTM Premix

ExPrime TaqTM DNA Polymerase

ExPrime TaqTM Premix

Ability Test of Products

Comparative Test of Products

Fidelity Test of Products

DNA Amplification Products

Keynote of Products

3GENET BIOㆍwww.genetbio.com

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▶Unique Compositions of Reaction Buffer

Relax the secondary structure of template

In case of using the general reaction buffer....

In case of using the GeNet Bio reaction buffer....

Denaturation/Annealing

PCR

PCR

Primer

Primer

A B M A B

M: DNA MarkerLane A: Company PLane B: GeNet Bio PCR enzymes

Improve the PCR yieldImprove the primer annealing

Denaturation/Annealing


Prime TaqTM DNA Polymerase

4 GENET BIOㆍwww.genetbio.com

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Feature▶General PCR enzyme▶Guarantees reproducible test result by its characteristic of high purity▶Amplifies target DNA by optimized buffer system▶Easily obtains under 5 Kb of DNA amplified products▶Prime TaqTM DNA Polymerase can be used in T-vector cloning

Description: Prime TaqTM DNA Polymerase is a high quality recombinant enzyme and catalyze 5'→3' synthesis of DNA.The enzyme has no detectable 3'→5' proofreading exonuclease activity. It is provided with 10X reaction buffer thatcontains PCR enhancers. This reaction buffer will enable or improve sub-optimal PCR caused by templates that havea high degree of secondary structure or that are GC-rich.

Cloning of PCR Products: A DNA fragment which is amplified by Prime TaqTM DNA Polymerase has A-overhang, andit enables you to do cloning by using T-vector.

Quality Control: Endonuclease, Exonuclease, DNase, RNase and Protease activity is not detected. Prime TaqTM DNA Polymerase is determined to be ＞90% pure as judged by SDS-PAGE.

Unit Definition: One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmoles ofdNTPs into acid-insoluble material in 30 minutes at 74℃.

Buffer and Reagents:Storage Buffer: 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM EDTA, 0.1 mM DTT, 0.5% Tween 20, 0.5 % Nonidet P-40, 50% Glycerol10X Reaction Buffer: Contains Tris-HCl (pH 9.0), PCR enhancers, (NH4)2SO4, 20 mM MgCl210 mM dNTPs Mixture: 2.5 mM each of dATP, dCTP, dGTP and dTTP

Cat. No.A-type

G-1000 250 units

G-1001 250 units X 2

G-1002 250 units X 4

B-type

G-1000-1 250 units

G-1001-1 250 units X 2

G-1002-1 250 units X 4

Conc. of Enzyme5 units/μl

Supplied withA-type

10X Reaction Buffer

(with 20 mM MgCl2)

with 10 mM dNTPs Mixture

B-type

10X Reaction Buffer

(with 20 mM MgCl2)

without dNTPs Mixture

StorageStore at -20℃


Prime TaqTM Premix


Prime

Taq

TM Prem

ix

Cat. No.G-2000 1 ml X 1

G-2001 1 ml X 3

G-2002 1 ml X 5

Feature▶Contains mixture of Prime TaqTM DNA Polymerase, dNTPs, reaction buffer in 2X concentration▶Contains loading dye and sediment for immediate loading to gel after reaction▶Provides the same result compare with manual formula▶Prime TaqTM Premix can be used in T-vector cloning

Description: Prime TaqTM Premix contains Prime TaqTM DNA Polymerase, reaction buffer, dNTPs mixture, protein stabilizer,and optimizes the convenience to use by adding sediment for electrophoresis and 2X solution of loading dye.In general, Prime TaqTM Premix shows no decline of activity compare with Prime TaqTM DNA Polymerase, even in a roomtemperature. Prime TaqTM Premix is good for under 5 Kb of PCR products, and also can obtain the result under 10 Kbby altering of some conditions (concentration of template, primer, and DNA Polymerase or increase of extension time).

Cloning of PCR Products: A DNA fragment which is amplified by Prime TaqTM Premix has A-overhang, and it enablesyou to do cloning by using T-vector.

Quality Control: Endonuclease, Exonuclease, DNase, RNase and Protease activity is not detected. Prime TaqTM DNA Polymerase is determined to be ＞90% pure as judged by SDS-PAGE.


Composition of 2X Premix Solution:Prime TaqTM DNA Polymerase 1 unit/10 μl, Tris-HCl (pH 9.0), PCR enhancer, (NH4)2SO4, 4 mM MgCl2, enzyme stabilizer,sediment, loading dye, 2.0 mM dNTPs mixture.


ExPrime TaqTM DNA Polymerase


ExPr

ime

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Feature▶Has the ability of 3'→5' exonuclease function (High Fidelity)▶Has an effect for DNA amplification of cloning and sequencing purpose▶Easily obtains under 10 Kb of DNA amplified products (Long PCR)▶Obtains high PCR yield compare with Prime TaqTM DNA Polymerase▶ExPrime TaqTM DNA Polymerase can be used in T-vector cloning

Description: ExPrime TaqTM DNA Polymerase has both 5'→3' exonuclease and 3'→5' exonucleas functions.Therefore, it has 3 times less error-rate compare with Taq DNA Polymerase, and it is efficient for cloning (High Fidelity).Also, it is easy to obtain PCR products in case of over 5 Kb as well as under 10 Kb of DNA amplified products (Long-PCR). Also ExPrime TaqTM DNA Poymerase gives you the satisfying result under 20 Kb by altering of some conditions(concentration of template, primer, DNA Polymerase or increase of extension time).

Cloning of PCR Products: A DNA fragment which is amplified by ExPrime TaqTM DNA Polymerase has A-overhang, andit enables you to do cloning by using T-vector.

Quality Control: Endonuclease, Exonuclease, DNase, RNase and Protease activity is not detected. ExPrime TaqTM DNA Polymerase is determined to be ＞90% pure as judged by SDS-PAGE.


Buffer and Reagents:Storage Buffer: 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.5 mM EDTA, 0.1 mM DTT, 0.5% Tween 20, 0.5 % Nonidet P-40, 50% Glycerol10X Reaction Buffer: Contains Tris-HCl (pH 9.0), PCR enhancers, (NH4)2SO4, 20 mM MgCl210 mM dNTPs Mixture: 2.5 mM each of dATP, dCTP, dGTP and dTTP

Cat. No.A-type

G-4000 250 units

G-4001 250 units X 2

G-4002 250 units X 4

B-type

G-4000-1 250 units

G-4001-1 250 units X 2

G-4002-1 250 units X 4

Conc. of Enzyme5 units/μl

Supplied withA-type

10X Reaction Buffer

(with 20 mM MgCl2)

with 10 mM dNTPs Mixture

B-type

10X Reaction Buffer

(with 20 mM MgCl2)

without dNTPs Mixture

StorageStore at -20℃


ExPrime TaqTM Premix


ExPrime

Taq

TM Prem

ix

Cat. No.G-5000 1 ml X 1

G-5001 1 ml X 3

G-5002 1 ml X 5

Feature▶Contains Mixture of ExPrime TaqTM DNA Polymerase, dNTPs, reaction buffer in 2X concentration▶Contains Loading dye and sediment for immediate loading to gel after reaction▶Provides the same result compare with manual formula▶ExPrime TaqTM Premix can be used in T-vector cloning

Description: ExPrime TaqTM Premix is composed of ExPrime TaqTM DNA Polymerse, reaction buffer, dNTP mixture, proteinstabilizer and sediment which is needed for electrophoresis, and 2X solution type of loading dye, and these componentsmaximize the convenience of the users. This product also has both 5→3 exonuclease and 3→5 exonucleas function thatExPrime TaqTM DNA Polymerase has. ExPrime TaqTM Premix shows no decline of activity compare with ExPrime TaqTM DNAPolymerase, even in a room temperature. Also, ExPrime TaqTM Premix gives you the satisfying result under 10 Kb as well asunder 20 Kb by altering of some conditions (concentration of template, primer, DNA Polymerase or increase of extensiontime).

Cloning of PCR Products: A DNA fragment which is amplified by ExPrime TaqTM Premix has A-overhang, and it enablesyou to do cloning by using T-vector.

Quality Control: Endonuclease, Exonuclease, DNase, RNase and Protease activity is not detected. ExPrime TaqTM DNA Polymerase is determined to be ＞90% pure as judged by SDS-PAGE.


Composition of 2X Premix Solution:ExPrime TaqTM DNA Polymerase 1 unit/10 μl, Tris-HCl (pH 9.0), PCR enhancer, (NH4)2SO4, 4 mM MgCl2, enzyme stabilizer,sediment, loading dye, 2.0 mM dNTPs mixture.



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▶Working Range Test

- Working range: Lambda DNA 0.5~20 Kb - PCR condition template: Lambda DNA 100 pg 94℃ 5min 94℃ 30sec, 58℃ 30sec, 72℃ 1min →30 cycles 72℃ 5min (15 Kb, 20 Kb size: 2 step PCR 94℃ 30sec, 68℃ 15min →30 cycles)

Prime TaqTM DNA Polymerase ExPrime TaqTM DNA Polymerase M1 1 2 3 4 5 6 7 8 9 M1 M1 1 2 3 4 5 6 7 8 9 10 11 M2

M1: 1 Kb DNA Ladder, M2: Lambda DNA/Hind III Markers lane 1: 0.5 Kb, lane 2: 1 Kb, lane 3: 2 Kb, lane 4: 3 Kb, lane 5: 4 Kb lane 6: 5 Kb, lane 7: 7 Kb, lane 8: 8 Kb, lane 9: 9 Kb, lane 10: 15 Kb lane 11:20 Kb

▶Sequence Specific PCR Test

- Target: Two polymorphisms of human IL-18 promoter site (position -607 and -137) -PCR products size Position -137: 446 bp, 261 bp Position -607: 301 bp, 196 bp

Prime TaqTM DNA Polymerase ExPrime TaqTM DNA Polymerase -137 -607 -137 -607 a M b c M d a M b c M d

M: 100 bp DNA Ladder a: Wild type (position -137), b: Hetero mutant (position -137) c: Homo mutant (position -607), d: Hetero mutant (position -607)



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▶HPV Detection Test

- Template: Extracted DNA from genetal sample - PCR condition 94℃ 5min 94℃ 30sec, 55℃ 30sec, 72℃ 30sec →35 cycles, 72℃ 5min 94℃ 5min 94℃ 30sec, 55℃ 30sec, 72℃ 30sec →21 cycles, 72℃ 5min

Prime TaqTM DNA Polymerase ExPrime TaqTM DNA Polymerase

M 1 2 3 4 M 1 2 3 4

M: 100 bp DNA Ladder lane 1: Negative lane 2: HPV type 16 lane 3: HPV type 31 lane 4: HPV type 35

1st PCR

2nd PCR (nested)

▶Genomic DNA Contamination Test

- This test is operated for the identification of E. coli genomic DNA contamination with positive (contain the E. coli genomic DNA) and negative (not contain the template) sample by PCR (30 ~ 50 cycles).

M C 1 2 3 C 1 2 3 C 1 2 3

A B C

Panel A: GeNet Bio Prime TaqTM DNA Polymerase Panel B: Company A standard Taq DNA Polymerase Panel C: Company P standard Taq DNA Polymerase

M: 100 bp DNA Ladder, C: control (positive) lane 1: 30 cycles, lane 2: 40 cycles, lane 3: 50 cycles



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Comparative Test of Products

▶HBV Detection Test from Blood, Saliva and Breast Milk Blood Saliva Breast milk

M: 1 Kb DNA Ladder

Lane 1: Prime TaqTM DNA Polymerase

Lane 2: Prime TaqTM Premix

Lane 3: ExPrime TaqTM DNA Polymerase

Lane 4: ExPrime TaqTM Premix

Lane 5: Company P

▶PCR Test from Hela cDNA Library

M 1 2 3 4 5 M 1 2 3 4 5 M 1 2 3 4 5 M

M 1 2 3 4 5 M

▶PCR Test from Fungi Genomic DNAM 1 2 3 4 5 6 M

M: 1 Kb DNA Ladder





Lane 5: Company P

M: 1 Kb DNA Ladder





Lane 5: Company P

Lane 6: Company T

→→

→



Fide

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Fidelity Test of Products

▶Fidelity of Prime TaqTM DNA Polymerase & ExPrime TaqTM DNA Polymerase

Relative Error Frquency

1

0.3

0.1

0

0.2

0.4

0.6

0.8

1

1.2

Prime TaqTM DNA Pol. ExPrime TaqTM DNA Pol. Pfu DNA Pol.

Enzyme

Rela

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* Reference1. Wayne M. Barnes The fidelity of Taq DNA Polymerase catalyzing PCR is improved by an N-terminal deletion. GENE 112(1992). 29-352. Wayne M. Barnes PCR amplification of up to 35-Kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91(1994). 2216-2220

Enzyme

Prime TaqTM

DNA Polymerase

ExPrime TaqTM

DNA Polymerase

Pfu DNAPolymerase

LacZ+

(blue colonies)

3,090

2,527

2,412

% Mutant

25.7

10.6

2.6

Errors Rate

7.34 X 10-5

3.04 X 10-5

0.73 X 10-5

Relative ErrorFrequency

1

0.4

0.1

LacZ-(white colonies)

1,068

301

64

GENET BIODankook Univ., Biotech Business Incubation Center B-123,

San 29, Anseo-dong, Cheonan-si, Chungnam, Korea, 330-714Tel.: +82-41-523-3484 Fax.: +82-41-523-3485

E-mail: [email protected] http://www.genetbio.com

N E W P A R A D I G M o f B I O T E C H N O L O G Y

NEW PARADIGM of BIOTECHNOLOGY -GENET BIOgenetbio.co.kr/FM/protein/Brochure.pdf · 2005. 9. 30. ·...

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