01 DNA Polymerase Selection Guide 293 Top DNA Polymerase 294 Taq DNA Polymerase 296 Pfu DNA Polymerase 298 ProFi Taq DNA Polymerase 300 HotStart DNA P

DNA Polymerase Reverse Transcriptase DNA Ligase Restriction Enzymes Enzymes Phone: 1588-9788 (ext.4->2) Email: reagents-support @bioneer.co.kr

01 DNA Polymerase Selection Guide 293 Top DNA Polymerase 294 Taq DNA Polymerase 296 Pfu DNA Polymerase 298 ProFi Taq DNA Polymerase 300 HotStart DNA Polymerase 302 HotStart Taq DNA Polymerase 304

Selection Guide Overview 바이오니아는 PCR, reverse transcription, DNA ligation의다양한실험에사용할수있는광범위한제품군을제공합니다. 가장기본적으로사용되는 Taq DNA polymerase와 HotStart 기능을가진 Hotstart DNA polymerase, proofreading을가진 Pfu polymerase등사용자의실험에맞게 enzyme선택이가능합니다. 바이오니아가보유한 thermostable DNA polymerase는기존에출시되어있는내열성효소중가장증폭속도가빠른효소입니다. 바이오니아의 CycleScript RTase( 특허등록 10-0984483, KOR) 는 55 에서도활성이유지되어고온에서도 cdna를합성할 수있고 2-3 단계의순차적인온도변화를통한순환온도역전사반응에이용할수있습니다. RocketScript TM RTase( 특허출원 10-110011639,KOR) 는 thermostable activity(42~70 ) 를갖고있어 complex RNA 의 full-length cdna 합성도효과적으로수행할수있는우수한성능의효소입니다. Selection Guide Choose the Bioneer Enzyme That s Right for You PCR RT Application 합성가격 DNA Polymerase HotStart DNA Polymerase Top Taq Pfu Top Taq ProFi Taq DNA Polymerase M-MLV RTase Reverse Transcriptase CycleScript RTase RocketScript TM RTase Standard PCR HotStart PCR High-fidelity PCR Long Kb PCR GC rich PCR Multiplex PCR Standard RT Cyclic RT Product Name Product Size Yield Specificity Fidelity GC-rich Heat Stability Top DNA Polymerase ~ 10 kb ***** **** *** *** *** Yes Taq DNA Polymerase ~ 10 kb ***** **** *** *** *** Yes HotStart Taq DNA Polymerase ~ 12 kb **** **** *** **** **** Yes HotStart DNA Polymerase ~ 12 kb **** **** *** **** **** Yes ProFi Taq DNA Polymerase ~ 30 kb **** **** *** **** **** Yes Pfu DNA polymerase ~ 10 kb ** *** **** ** *** No M-MLV Reverse Transcriptase ~ 9 kb *** - - *** ** - CycleScript Reverse Transcriptase ~ 9 kb **** - - *** *** - RocketScript TM Reverse Transcriptase ~ 10 kb **** - - *** **** - Leaves 3'-A www.bioneer.co.kr 293

Top DNA Polymerase Enzymes for Everyday PCR, Faster than Taq DNA Polymerase, and TA Cloning Compatible Application Real-Time quantification of DNA and cdna targets using SYBR Green dye. Gene expression profiling Microbial & viral pathogen detection Reagents Supplied 10 x Reaction buffer with (or without) MgCl 2: 100 mm Tris- HCl, 400 mm KCl, 15 mm MgCl 2, ph 9.0 1 x Dilution buffer: 50 mm Tris-HCl, 0.1 mm EDTA, 1 mm DTT, stabilizers, 50 % Glycerol ph 8.0 dntps mixture: 10 mm, each dntp 2.5 mm Description Top DNA polymerase 는 Thermophile polymerase gene 을 DNA recombination 기술을이용하여 polymerase activity 를향상시킨후 cloning 하여, E. coli 에발현및고순도로정제한 94 kda 의 thermostable DNA polymerase 입니다. Top DNA polymerase 의 quality 는 activity test, SDS-PAGE / purity test, endonuclease /nickase test 를수행하여확인하였고, PCR 방법을사용한다양한 DNA 증폭실험에사용할수있습니다. Features and Benefits High yields: Taq DNA polymerase 에비해향상된증폭효율을갖습니다. High sensitivity: Taq DNA polymerase 에비해향상된민감도를갖습니다. Stable reaction: 최적화된버퍼를제공하여 Top DNA polymerase 의안정적반응을보증합니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Concentration 500 U (5 U/μl) Storage Conditions 50 mm Tris-HCl, 0.1 mm EDTA, 1 mm DTT, stabilizers, 50% Glycerol ph 8.0 Storage Temperature -20 Unit Definition One is defined at the amount of enzyme that will incorporate 10 nmole of dntp into acid-insoluble material in 30 min at 72. Specifications 5 to 3 exonuclease activity: No 3 to 5 exonuclease activity: No 3 -A overhang: Yes Fragment Size: ~10 kb BIONEER 294

Top DNA Polymerase Experimental Data Top DNA Polymerase Taq DNA Polymerase 1 2 3 4 5 6 7 8 MM 1 2 3 4 5 6 7 8 Figure 1. Enzyme activity test of Top DNA polymerase and Taq DNA polymerase. Top DNA polymerase/ Taq DNA polymerase was serially diluted and used to amplify 20 ng of each lambda and human genomic DNA. Lane 1: 1 U of Top DNA Polymerase used Lane 2: 0.5 U of Top DNA Polymerase used Lane 3: 0.33 U of Top DNA Polymerase used Lane 4: 0.25 U of Top DNA Polymerase used Lane 5: 1 U of Taq DNA Polymerase used Lane 6: 0.5 U of Taq DNA Polymerase used Lane 7: 0.33 U of Taq DNA Polymerase used Lane 8: 0.25 U of Taq DNA Polymerase used Lane MW: 100 bp plus DNA Ladder (Bioneer, D-1035) M1 M2 1 2 3 4 5 6 7 8 9 M1 M2 1 2 3 4 5 6 7 8 9 Figure 2. Long PCR amplification test of Top DNA Polymerase and Taq DNA Polymerase using Lambda DNA. 10 ng of Lambda DNA and 1 U of each DNA Polymerase used for amplification. Lane 1: 2 kb PCR product Lane 2: 3 kb PCR product Lane 3: 4 kb PCR product Lane 4: 5 kb PCR product Lane 5: 6 kb PCR product Lane 6: 7 kb PCR product Lane 7: 8 kb PCR product Lane 8: 9 kb PCR product Lane 9: 10 kb PCR product M1: 1 kb DNA Ladder (Bioneer, D-1040) M2: Lambda DNA/Hind lll Marker (Bioneer, D-1050) Figure 3. Sensitivity test using Real-Time qpcr with SYBR Green and Top DNA Polymerase. The standard curve shows a high correlation of R 2 = 0.9993. Lane 1: 100 ng lambda DNA Lane 2: 10 ng lambda DNA Lane 3: 1 ng lambda DNA Lane 4: 100 pg lambda DNA Lane 5: 10 pg lambda DNA Lane 6: 1 pg lambda DNA Lane 7: 100 fg lambda DNA Lane 8: 10 pg lambda DNA Note: 본제품에포함된 Top DNA polymerase 는 base incorporation rate 를증가시키기위하여 point mutation 을통해 5 ->3 exonuclease 활성을없앤효소입니다. 그러므로 SYBR green 을이용한 Real-Time qpcr 에는사용하실수있지만, TaqMan probe 등의 probe 를이용하는 Real-Time PCR 에는사용하실수없습니다. TaqMan probe 를이용한 qpcr 실험을위하여 AccuPower Dualstar TM qpcr PreMix 제품을권장합니다. Ordering Information Product Description D-3001 10 mm dntps Mix (each 2.5 mm), 1 ml E-3100 Top DNA Polymerase, 500 U, 10 mm dntps, 10 x reaction buffer with MgCl 2 E-3100-1 Top DNA Polymerase, 500 U, 10 mm dntps, 10 x reaction buffer, 20 mm MgCl 2 E-3100-2 Top DNA Polymerase, 500 U, 10 x reaction buffer with MgCl 2 E-3100-3 Top DNA Polymerase, 500 U, 10 x reaction buffer, 20 mm MgCl 2 E-3101 Top DNA Polymerase, 2,000 U, 10 mm dntps, 10 x reaction buffer with MgCl 2 E-3101-1 Top DNA Polymerase, 2,000 U, 10 mm dntps, 10 x reaction buffer, 20 mm MgCl 2 E-3101-2 Top DNA Polymerase, 2,000 U, 10 x reaction buffer with MgCl 2 E-3101-3 Top DNA Polymerase, 2,000 U, 10 x reaction buffer, 20 mm MgCl 2 www.bioneer.co.kr 295

Taq DNA Polymerase Versatile DNA Polymerase for Everyday Routine PCR Application Real-Time quantification of DNA and cdna targets using Dual probe, SYBR Green dye Gene expression profiling Microbial & viral pathogen detection Reagents Supplied 10 x Reaction buffer with (or without) MgCl 2: 100 mm Tris- HCl, 400 mm KCl, 15 mm MgCl 2, ph 9.0 Dilution buffer: 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm DTT, 100 mm KCl, Stablizers, 50 % Glycerol, ph 8.0 dntps mixture: 10 mm, each dntp 2.5 mm Description Taq DNA Polymerase 는 Thermus aquaticus YT1 유래 DNA polymerase gene 을재조합기술을이용하여 Escherichia coli 로부터발현시킨후순수분리정제된 94 kda 의내열성 Thermus aquaticus DNA polymerase 입니다. Features and Benefits High yields & high sensitivity: 높은증폭효율과 template 에대한높은민감도를갖습니다. Equipment Compatibility: 여러종류의실시간 PCR 장비에최적화된결과를얻을수있습니다. Stable reaction: 최적화된버퍼를제공하여 Taq DNA Polymerase 의안정적반응을보증합니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다 Concentration 500 U (5 U/μl) Storage Conditions 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm DTT, 100 mm KCl, Stablizers, 50 % Glycerol ph 8.0 Storage Temperature -20 Unit Definition One is defined at the amount of enzyme that will incorporate 10 nmole of dntp into acid-insoluble material in 30 min at 72. Specifications 5 to 3 exonuclease activity: Yes 3 to 5 exonuclease activity: No 3 - A overhang: Yes Fragment size: ~10 kb BIONEER 296

Taq DNA Polymerase Experimental Data (A) (B) M 1 2 3 4 5 1 2 3 4 5 6 7 Figure 1. Using 1 of Taq DNA polymerase, the activity of the polymerase was tested on human genomic DNA (A), lambda genomic DNA (B) as template. Each template DNA was serially diluted by tenfolds, with different ranges. Lane 1: 100 ng Template DNA Lane 2: 10 ng Template DNA Lane 3: 1 ng Template DNA Lane 4: 100 pg Template DNA Lane 5: 10 pg Template DNA Lane 6: 1 pg Template DNA Lane 7: 100 fg Template DNA M: 1 kb DNA Ladder (Bioneer, D-1040) Figure 2. Amplification of fragments ranging from 5 kb to 8 kb from template Lambda DNA 20 pg with 1 s of Taq DNA Polymerase. Lane 1: 5 kb PCR product Lane 2: 6 kb PCR product Lane 3: 7 kb PCR product Lane 4: 8 kb PCR product M: 1 kb DNA Ladder (Bioneer, D-1040) M 1 2 3 4 Ordering Information Product Description D-3001 10 mm dntps Mix (each 2.5 mm), 1 ml E-2011 Taq DNA Polymerase, 500 U, 10 mm dntps, 10 x reaction buffer with MgCl 2 E-2011-1 Taq DNA Polymerase, 500 U, 10 mm dntps, 10 x reaction buffer, 20 mm MgCl 2 E-2011-2 Taq DNA Polymerase, 500 U, 10 x reaction buffer with MgCl 2 E-2011-3 Taq DNA Polymerase, 500 U, 10 x reaction buffer, 20 mm MgCl 2 E-2013 Taq DNA Polymerase, 2,000 U, 10 mm dntps, 10 x reaction buffer with MgCl 2 E-2013-1 Taq DNA Polymerase, 2,000 U, 10 mm dntps, 10 x reaction buffer, 20 mm MgCl 2 E-2013-2 Taq DNA Polymerase, 2,000 U, 10 x reaction buffer with MgCl 2 E-2013-3 Taq DNA Polymerase, 2,000 U, 10 x reaction buffer, 20 mm MgCl 2 www.bioneer.co.kr 297

Pfu DNA Polymerase Novel Enzyme for High Fidelity PCR with DNA Proofreading Specifications 5 to 3 exonuclease activity: No 3 to 5 exonuclease activity: Yes 3 -A overhang: No Fragment size: ~10 kb Application Gene synthesis PCR or Primer extension requested High fidelity Blunt-end PCR Cloning or mutagenesis requested high fidelity Description Pfu DNA Polymerase 는 Pyrococus furiosus 라는 bacteria 에서유래되었으며, 3 5 exonuclease (proofreading) activity 를지니고있어기존의 Taq DNA Polymerase 보다 heat stability 와 fidelity 가뛰어납니다. 또한뛰어난 specificity 를가지고있어 nonspecific product 의발생이적으며, proofreading 기능이있어 DNA 증폭시발생하는 error rate 을감소시킵니다. Pfu DNA Polymerase 는 gene cloning, PCR, primer extension 등다양한실험에사용하실수있습니다. Features and Benefits High fidelity PCR: 3 5 proofreading activity 가뛰어납니다. Thermostability: 열안정성이뛰어나 95 에서 1 시간반응후에도 94~99% 의기능을유지합니다. Terminal Transferase Activity: terminal transferase activity 가없어 blunt-ended PCR product 를얻을수있습니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Reagents Supplied 10 x Reaction Buffer: 300 mm Tris-HCl, 200 mm KCl, 100mM (NH 4) 2SO 4, 15 mm MgSO 4, Acetylated BSA, ph 9.0 Dilution buffer: 50 mm Tris-HCl, 0.1 mm EDTA, 1 mm DTT, Stablizers, 50 % Glycerol, ph 8.0 dntps mixture: 10 mm, each dntp 2.5 mm (optional) Concentration 250 U (2.5 U/μl) Storage Temperature -20 Unit Definition One is defined at the amount of enzyme that will incorporate 10 nmol of dntp into acid-insoluble material in 30 min at 72. BIONEER 298

Pfu DNA Polymerase Experimental Data M 1 2 3 4 M 1 2 3 4 Figure 1. Human DNA was amplified using 2.5 s of enzyme in 50 μl reaction volume. Lane 1: 20 ng Lane 2: 2 ng Lane 3: 200 pg Lane 4: 20 pg M: 100 bp DNA ladder(bioneer, D-1030) Figure 2. Lambda DNA 를이용한 Pfu DNA polymerase 의 Long kb PCR test. Lane 1: Lambda DNA 5 kb Lane 2: Lambda DNA 6 kb Lane 3: Lambda DNA 7 kb Lane 4: Lambda DNA 8 kb M: 1 kb DNA ladder(bioneer, D-1040) Ordering Information Product Description E-2015 Pfu DNA Polymerase, 250 U, 10 x reaction buffer, without dntps E-2015-1 Pfu DNA Polymerase, 250 U, 10 x reaction buffer, 10 mm dntps E-2016 Pfu DNA Polymerase, 1,000 U, 10 x reaction buffer, without dntps www.bioneer.co.kr 299

ProFi Taq DNA Polymerase For High Efficiency and Amplification of Long Range PCR. Specifications 5 to 3 exonuclease activity: Yes 3 to 5 exonuclease activity: Yes 3 -A overhang: Yes PCR product size: ~30 kb Application Primer extension long-range amplification from genomic DNA High amplification efficiency Excellent performance on difficult templates Description ProFi Taq DNA polymerase 는합성생물학기술을이용하여개발한 recombinant Taq DNA polymerase 로써뛰어난증폭효율과정확성을동시에충족시키는 DNA polymerase 입니다. ProFi Taq DNA polymerase 는 Taq DNA polymerase 를개선한효소로써기존의 Taq DNA polymerase 와비교하여뛰어난증폭성능, 높은 efficiency 를가지는제품입니다. ProFi Taq DNA polymerase 는 human genomic DNA 의경우 21 kb 까지 DNA fragment 증폭이가능하며 lambda DNA 의경우 30 kb 까지 DNA fragment 증폭이가능합니다. ProFi Taq DNA polymerase 는 high efficiency 및 long-range PCR 에매우적합한제품이며그외에도 complex genomic DNA 또는 cdna templates, low-copy targets 등다양한 PCR 증폭반응에사용하실수있습니다. Features and Benefits Sensitivity: Lambda DNA (100 ng~10 fg) 및 human genomic DNA (100 ng~100 pg) 를 template 로한타사비교실험결과, 타사제품에비해좋은증폭효율및민감도를가집니다. Long PCR: Lambda DNA 의경우 30 kb, human genomic DNA 의경우 21 kb 의 PCR 산물을효과적으로증폭시킬수있습니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Amplification of low-copy targets High yield and high sensitivity PCR Reagents Supplied 10 x Reaction Buffer: 400 mm Tris-HCl, 600 mm KCl, 15 mm MgCl 2, Acetylated BSA, ph 9.0 Dilution buffer: 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm DTT, 100 mm KCl, Stablizers, 50 % Glycerol, ph 8.0 dntps mixture: 10 mm, each dntp 2.5 mm Concentration 250 U (5 U/μl) Storage Temperature -20 BIONEER 300

ProFi Taq DNA Polymerase Experimental data Bioneer M 1 2 3 4 Supplier T 1 2 3 4 Supplier S 1 2 3 4 Supplier I 1 2 3 4 Figure 1. Comparison of PCR amplification efficiency between ProFi Taq DNA polymerase from Bioneer and other suppliers DNA polymerase. The cycling conditions for ProFi Taq DNA polymerase were 95 for 5 min, 30 cycles of 95 for 20 sec, 55 for 20 sec and 72 for 30 sec. PCR reaction using other suppliers DNA polymerase were performed according to each supplier s protocol. Target: human Insulin receptor gene Lane 1: 10 ng of human genomic DNA Lane 2: 1 ng of human genomic DNA Lane 3: 100 pg of human genomic DNA Lane 4: 10 pg of human genomic DNA Lane M: 100 bp DNA Ladder(Bioneer, D-1030) Bioneer M1 M2 1 2 3 4 Supplier I 1 2 3 4 Supplier S 1 2 3 4 Supplier T 1 2 3 4 M2 M1 Figure 2. Comparison of PCR amplification of long targets between ProFi Taq DNA polymerase from Bioneer and other suppliers DNA polymerase The cycling conditions for ProFi Taq DNA polymerase were 95 for 5 min, 32 cycles of 95 for 20 sec and 68 for 15 min. PCR reactions using other suppliers DNA polymerase were performed according to each supplier s protocol. Human genomic DNA was used as a template for PCR amplification. Lane 1: 11 kb fragment Lane 2: 13.5 kb fragment Lane 3: 17.6 kb fragment Lane 4: 21.4 kb fragment Lane M1: Lambda/Hind III marker (Bioneer, D-1050) Lane M2: 1 kb DNA Ladder (Bioneer, D-1040) Figure 3. Comparison of PCR amplification of long targets between ProFi Taq DNA polymerase from Bioneer and other suppliers DNA polymerase. The cycling conditions for ProFi Taq DNA polymerase were 95 for 5 min, 30 cycles of 95 for 20 sec, 65 for 20 sec and 68 for 4 min. PCR reactions using other suppliers DNA polymerase were performed according to each supplier s protocol. Lane 1: 2 kb fragment (human tumor protein p53 gene) Lane 2: 3 kb fragment (human tumor protein p53 gene) Lane 3: 4.5kb fragment (human DNA cross-link repair 1A gene) Lane 4: 8 kb fragment (human hemoglobin epsilon 1 gene) Lane M1: Lambda/Hind III marker (Bioneer, D-1050) Lane M2: 1 kb DNA Ladder (Bioneer, D-1040) Ordering Information Product Description E-2201 ProFi Taq DNA Polymerase 250 U, 10 mm dntps, 10 X reaction buffer with MgCl 2 E-2202 ProFi Taq DNA Polymerase 250 U, 10 mm dntps, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2 E-2203 ProFi Taq DNA Polymerase 250 U, 10 X reaction buffer with MgCl 2 E-2204 ProFi Taq DNA Polymerase 250 U, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2 E-2205 ProFi Taq DNA Polymerase 1000 U, 10 mm dntps, 10 X reaction buffer with MgCl 2 E-2206 ProFi Taq DNA Polymerase 1000 U, 10 mm dntps, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2 E-2207 ProFi Taq DNA Polymerase 1000 U, 10 X reaction buffer with MgCl 2 E-2208 ProFi Taq DNA Polymerase 1000 U, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2 www.bioneer.co.kr 301

HotStart DNA Polymerase Unique Enzyme-Mediated HotStart DNA Polymerase Features and Benefits High Sensitivity and High Specificity: 상온에서의 DNA polymerase 활성을완벽하게억제하였기때문에 PCR 반응물을혼합하는동안형성될수있는 non-specific band 나 primerdimer 의생성을억제합니다. Improve product yields: 높은 sensitivity 와 specificity 로인해원하는증폭산물의 yield 를향상시킬수있습니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Specifications 5 to 3 exonuclease activity: No Description 항체를사용하여 Polymerase 를불활성화시켰던기존의 HotStart 방법과는달리, PCR 반응에필수적인 2 가양이온 (Mg 2+ ) 을불활성화시키는피로포스페이트 (pyrophosphate, PPi) 와 70 에서활성을갖는내열성 pyrophosphatase(ppase) 를사용한신개념의 HotStart DNA polymerase 입니다. New Concept by Chemical Method Pyrophosphate(PPi) 와 Pyrophosphatase(PPase) 의화학반응을이용한신개념 HotStart DNA Polymerase. 3 to 5 exonuclease activity: No 3 -A overhang: Yes Fragment size: ~12 kb Application HotStart PCR, PCR with complex genomic templates/low copy templates/cdna Multiplex PCR Primer extension SNP typing PCR 반응의 General Equation Template + Primer + Mg 2+ +dntp Elongation + 2Pi Real-Time PCR using SYBR Green dye Multiple primer pairs and amplification of low copy template DNA 일반적인 PCR 반응성분중에서 Mg 2+ 는 DNA polymerase 의 activity 에중요한역할을합니다. Mg 2+ 와결합력이강한 PPi 이용하여 PCR 초기단계에 Mg 2+ 의기능을억제하면비특이적증폭을최소화할수있습니다. PCR 반응액에 PPase 와 PPi 를넣어주면 PPi 는 Mg 2+ 와결합하여 Mg 2+ -PPi complex 를형성하고 PCR 반응을억제함으로써 PCR 초기단계에서발생하는비특이반응을방지합니다. 그후 PCR 반응액이 70 이상이되면 PPase 가활성화되어 PPi 를 2Pi 로분해시키고 Mg 2+ 와해리됨으로써정상적인 PCR 반응이진행됩니다. 또한반응부산물이면서동시에 PCR 저해제인 PPi 를 PPase 가 2Pi 로분해함으로써 PCR 반응성을증가시킵니다. 무엇보다항체를사용하지않기때문에항체제거 step 이필요치않아 PCR 반응시간을단축시키는장점을가지고있습니다. Reagents Supplied 10 x Reaction buffer: 100 mm Tris-HCl, 400 mm KCl, 20 mm Pyrophosphate, ph 9.0 1 x Dilution buffer: 50 mm Tris-HCl, 0.1 mm EDTA, 1 mm DTT, stabilizers, 50% Glycerol, ph 8.2 dntps mixture: 10 mm, each dntp 2.5 mm 20 mm MgCl 2 Concentration 250 U (5 U/μl) BIONEER 302

HotStart DNA Polymerase Storage Conditions 50 mm Tris-HCl, 0.1 mm EDTA, 1 mm DTT, stabilizers, 50% Glycerol, ph 8.2 Storage Temperature -20 Unit Definition One is defined at the amount of enzyme that will incorporate 10 nmole of dntp into acid-insoluble material in 30 min at 72. Experimental Data Figure 1. Multiplex PCR comparison of genomic DNA using 6 sets of primers and 2 different DNA Polymerases. Lane 1: 750 bp fragment Lane 2: 590 bp fragment Lane 3: 450 bp fragment Lane 4: 360 bp fragment Lane 5: 260 bp fragment Lane 6: 150 bp fragment Lane 7: Multiplex PCR with primers used for Lane 1~6 Lane M: 100 bp DNA Ladder (Bioneer, D-1030) Ordering Information Product Description E-3150 HotStart DNA Polymerase, 250 U, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2, 10 mm dntps E-3150-1 HotStart DNA Polymerase, 250 U, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2 E-3151 HotStart DNA Polymerase, 1,000 U, 10 X reaction buffer without MgCl 2, 20 mm MgCl 2, 10 mm dntps www.bioneer.co.kr 303

HotStart Taq DNA Polymerase For Increased Specificity and Robust Sensitivity. Reagents Supplied 10X Reaction Buffer: 100 mm Tris-HCl, 450 mm KCl, 15 mm MgCl 2, ph 9.0 Dilution Buffer: 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm DTT, 100 mm KCl, Stabilizers, 50 % Glycerol, ph 8.0 dntps mixture: 10 mm, each dntp 2.5 mm Concentration 250 U (5 U/μl) Description HotStart Taq DNA polymerase 는상온에서는활성이없다가 95 에서활성을가지게함으로써특이성이높은 PCR 산물을만들어낼수있도록개발되었습니다. 일반적인 PCR 반응시상온에서 PCR 반응물들을섞을때부터발생하는 mispriming 이나 primer 의 dimer 형성에의한비특이적인증폭을방지할수있습니다. Features and Benefits High Specificity & Fidelity: 일반적인 Taq DNA polymerase 나 PCR premix 보다우수한결과를얻을수있습니다. High sensitivity: 불필요한반응이감소됨으로써소량의 template 양으로도 PCR 수행이가능합니다. Easy Handling: 상온에서 polymerase 가활성을띄지않으므로 PCR 반응물을섞을때비특이적 Priming 을방지할수있습니다. Simplicity: PCR 산물은 3 말단에 A tail 을가지고있어 T vector 에바로 cloning 할수있습니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Specifications 5 to 3 exonuclease activity: Yes 3 to 5 exonuclease activity: No 3 -A overhang: Yes Fragment size: ~10 kb Application Real-Time quantification of DNA and cdna targets using SYBR Green dye. HotStart PCR Multiplex PCR Automated PCR Storage Conditions 20 mm Tris-HCl, 0.5 mm EDTA, 1 mm DTT, 100 mm KCl, Stabilizers, 50 % Glycerol, ph 8.0 Storage Temperature -20 Unit Definition One is defined at the amount of enzyme that will incorporate 10 nmole of dntp into acid-insoluble material in 30 min at 72. Experimental Data General thermostable DNA polymerase HotStart Taq DNA polymerase of Bioneer M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8 Figure 1. Specificity comparison between standard Taq and HotStart Taq DNA polymerase. Single and multiples PCR results in human genomic DNA p53 Gene amplification Lane 1: 139 bp Lane 2: 211 bp Lane 3: 447 bp Lane 4: 618 bp Lane 5: 1,082 bp Lane 6: 1,296 bp Lane 7: 1,561 bp Lane 8: Multiplex PCR(139 bp, 447 bp, 618 bp) M: 100 bp DNA Ladder (Bioneer, Cat. no D-1030) BIONEER 304

HotStart Taq DNA Polymerase HotStart Taq DNA polymerase of Bioneer HotStart DNA polymerase HotStart DNA polymerase M A B Figure 2. Performance comparison between HotStart Taq polymerase and other supplier. M: 100 bp DNA Ladder (Bioneer, D-1040) A: competitor A B: competitor B Ordering Information Product Description E-2017 HotStart Taq DNA Polymerase, 250 U, 10 x reaction buffer with MgCl 2, 10 mm dntps E-2017-1 E-2017-2 HotStart Taq DNA Polymerase, 1,000 U, 10 x reaction buffer with MgCl 2, 10 mm dntps HotStart Taq DNA Polymerase, 500 U, 10 x reaction buffer with MgCl 2, 10 mm dntps E-2017-3 HotStart Taq DNA Polymerase, 250 U, 10 x reaction buffer with MgCl 2 E-2017-4 HotStart Taq DNA Polymerase, 1,000 U, 10 x reaction buffer with MgCl 2 www.bioneer.co.kr 305

02 Reverse Transcriptase M-MLV Reverse Transcriptase 307 CycleScript Reverse Transcriptase 308 RocketScript Reverse Transcriptase 311

M-MLV Reverse Transcriptase Standard cdna Synthesis Specifications 5 to 3 exonuclease activity: No 3 to 5 exonuclease activity: No 3 -A overhang: No Strand displacement: Yes Fragment size: ~9 kb Application First strand cdna synthesis from RNA, RT-PCR, and qrt-pcr Description Moloney Murine Leukemia Virus (M-MLV) 에서유래되었으며, RNA dependent DNA polymerase 로써 RNA 를주형으로 firststrand cdna 를합성할수있습니다 Source: M-MLV Reverse Transcriptase is isolated from an E. coli strain containing a recombinant clone. Features and Benefits Optimized 5X buffer: cdna 합성이 10 분이내에가능합니다. Full length cdna: 최대 9 kb 까지합성이가능합니다. RNase, DNase and Proteinase-free: RNase, DNase, Proteinase 활성을제거하여 deletion type 의 RTase M-MLV 에비해합성능력이우수합니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Reagents Supplied 5 x Reaction Buffer: 150 mm Tris-HCl, 250 mm KCl, 10 mm MgCl 2, ph 8.1 100 mm DTT dntps mixture: 10 mm, each dntp 2.5 mm Concentration 10,000 U (200 U/μl) Storage Conditions 20 mm Tris-Cl, 150 mm NaCl, 0.1 mm EDTA, 1 mm DTT, 0.1 % IGEPAL CA-630, 50% Glycerol, ph 7.6 Storage Temperature -20 Unit Definition One is defined as the amount of enzyme required to incorporates 1 nmole of dttp into acid-precipitable material in 10 min at 37 using poly A, oligo dt as template primer. Ordering Information Product Description E-3121 M-MLV Reverse Transcriptase, 10,000 U, 5 x Reaction Buffer, 100 mm DTT, 10 mm dntps E-3122 M-MLV Reverse Transcriptase, 50,000 U, 5 x Reaction Buffer, 100 mm DTT, 10 mm dntps www.bioneer.co.kr 307

CycleScript Reverse Transcriptase High Performance cdna Synthesis Description CycleScript Reverse Transcriptase (CTase) 는고온에서도 cdna 를합성할수있는제품으로고순도로정제된역전사효소에안정화물질을첨가하였습니다. 본제품은기존의 42 단일온도역전사반응 (Fixed Temperature Reverse Transcription, FTRT) 뿐만아니라, PCR 반응과같이 2-3 단계의순차적인온도변화를통한순환온도역전사반응 (Cyclic Temperature Reverse Transcription [CTRT], patent pending) 을수행할수있는제품입니다. 상기단일온도역전사반응 (FTRT) 및순환적역전사반응 (CTRT) 과정은다음과같습니다. 상기단일온도역전사반응 (FTRT) 및순환적역전사반응 (CTRT) 과정은다음과같습니다. 단일온도역전사반응 (FTRT) - Step 1: RNA denaturation 단계 (65, 10 min) - Step 2: cdna 합성단계 (42, 15~60 min) 순환온도역전사반응 (CTRT) - Step 1: Primer annealing 단계 (25~40, 30 sec) - Step 2: cdna 합성단계 (42~48, 4 min) - Step 3 (optional): RNA template 2 차구조해소및 cdna 합성단계 (50~55, 30 sec) * 상기반응에서온도, 시간및 cycle 은고객의실험조건에따라선택적으로설정할수있습니다. 바이오니아가특허출원한순환온도역전사반응은저온반응 (15~40 ) 에서 primer annealing 을유도하고, 고온반응 (50~55 ) 에서 template RNA 이차구조형성을해소함으로써기존의 42 역전사반응보다효율적으로 cdna 를합성할수있습니다. 또한, 역전사반응에필요한 DTT, CycleScript Reverse Transcriptase, reaction buffer 외에 dntps mixture 도포함하고있어별도의구매가필요하지않습니다. Features and Benefits Stability: RNase, DNase & Proteinase free 한순수한 reverse transcriptase (RTase, M-MLV) 에안정화물질을첨가하여효소의열안정성을높였습니다. High cdna synthesis efficiency: 저온반응에서 primer annealing 을유도하고, 고온에서 RNA template 의 2 차구조를해소하여 cdna 합성율의증가시키고 full length cdna 합성을가능하게합니다. Flexible reaction conditions: CycleScript Reverse Transcriptase 는단일온도에서의역전사반응뿐만아니라순환온도역전사반응에적용할수있도록열안정성을높인제품입니다. Ease-of-use & Speed: 순환적역전사반응을수행할경우 primer 와 RNA template 의 pre-incubation 과정이생략되어실험방법이간단하고반응시간이단축됩니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Specifications 5 to 3 exonuclease activity: No 3 to 5 exonuclease activity: No 3 -A overhang: Yes Strand displacement: Yes Fragment size: ~9 kb BIONEER 308

CycleScript Reverse Transcriptase Application First-strand synthesis of cdna from RNA molecules RT-PCR Random priming reaction Library construction Probe labeling mrna 5 end mapping by primer extension analysis Reagents Supplied 5 x Reaction Buffer: 150 mm Tris, 250 mm KCl, 10 mm MgCl 2, ph 8.1 100 mm DTT dntps mixture: 10 mm, each dntp 2.5 mm Concentration 10,000 U (200 U/μl) Storage Conditions 20 mm Tris-Cl, 150 mm NaCl, 0.1 mm EDTA, 1 mm DTT, 0.1% IGEPAL CA-630, 50% Glycerol, ph 7.6 Storage Temperature -20 Unit Definition One is defined as the amount of enzyme required to incorporates 1 nmole of dttp into acid-precipitable material in 10 min at 37 using poly A oligo dt as template primer. Principles 일반적으로사용되는 oligo dt 및 random primer 는 Tm 값이 15~40 정도로낮아기존의 42 역전사반응에서는 template RNA 와충분히 annealing 하기어렵고, cdna 합성에장애가되는 template RNA 2 차구조가유지되는경향이있습니다. 바이오니아의순환온도역전사반응 ( 특허출원중 ) 은저온반응 (15~40 ) 에서 primer annealing 을유도하고고온반응 (50~55 ) 에서 template RNA 이차구조형성을해소함으로써효율적으로 cdna 를합성할수있습니다. www.bioneer.co.kr 309

CycleScript Reverse Transcriptase Experimental Data Figure 1. Comparison of transferrin receptor gene amplification with different reverse transcriptases. 700 ng of total RNA was used for reverse transcription and the same amount of amplified products were used for electrophoresis. Lane 1-4: TFR (Transferrin receptor gene) amplified with M-MLV Lane 5-8: TFR amplified with CycleScript Lane 9-12: TFR amplified with CycleScript Lane 13-16: TFR amplified with M-MLV from Company I Lane 17-20: TFR amplified with S-script from Company S Lane 21-24: TFR amplified with S-script ll from Company I Lane 25-28: TFR amplified with S-script lll from Company I Lane 29-32: TFR amplified with O-script from Company Q Lane M.W.: 100 bp Plus DNA Ladder (Bioneer, D-1035) Figure 2. Comparison of GAPDH gene amplification with different reverse transcriptases. Each 10 ng, 1 ng, 100 pg, and 10 pg of total RNA was used for reverse transcription and the same amount of amplified products were used for electrophoresis. Lane 1-4: GAPDH amplified with CycleScript Lane 5-8: GAPDH amplified with CycleScript Lane 9-12: GAPDH amplified with CycleScript Lane 13-16: GAPDH amplified with M-MLV from Company I Lane 17-20: GAPDH amplified with S-script from Company S Lane 21-24: GAPDH amplified with S-script from Company S Lane 25-28: GAPDH amplified with S-script from Company I Lane 29-32: GAPDH amplified with S-script from Company I Figure 3. Working temperature comparison of different reverse transcriptases. Each 10 ng, 1 ng, 100 pg, and 10 pg of total RNA was used for reverse transcription and the same amount of amplified products were used for electrophoresis. Lane 1-4: GAPDH amplified with CycleScript Lane 5-8: GAPDH amplified with CycleScript Lane 9-12: GAPDH amplified with CycleScript Lane 13-16: GAPDH amplified with S-script from company S Lane 17-20: GAPDH amplified with S-script from company S Lane 21-24: GAPDH amplified with S-script from company S Ordering Information Product Description E-3131 CycleScript Reverse Transcriptase, 10,000 U, 5 x Reaction Buffer, 100 mm DTT, 10 mm dntps E-3132 CycleScript Reverse Transcriptase, 50,000 U, 5 x Reaction Buffer, 100 mm DTT, 10 mm dntps BIONEER 310

RocketScript Reverse Transcriptase High Performance / High Temperature cdna Synthesis Specifications 5 to 3 exonuclease: No 3 to 5 exonuclease: No 3 -A overhang: No Fragment size: ~10 kb Description Rocketscript TM Reverse Transcriptase 는바이오니아가독자적으로개발한 M-MLV 유래의 Thermostable Reverse Transcriptase 입니다. 저온 (42 ) 에서최적활성을갖는 M-MLV RTase 는복잡한 2 차구조를가진 RNA 로부터 cdna 를합성하는데많은문제점이있습니다. Rocketscript TM RTase 는 thermostable activity(42~70 ) 를갖고있어 complex 구조의 RNA 로부터효과적으로 full-length cdna 를합성할수있습니다. Features and Benefits Thermostable Activity: M-MLV RTase 효소의경우낮은열안정성을가지며, 이에따라역전사반응은낮은온도 (42 ) 에서만역전사반응이진행됩니다. 때문에복잡한고차구조의 RNA 를 cdna 로합성하는경우반응이제대로진행되지않는단점이있습니다. 이러한문제점을해결하기위해바이오니아는 synthetic biology 기술을이용하여 50 이상의높은온도에서도활성을갖는 Reverse Transcriptase 를개발하였습니다. 기존 42 RT 반응에만국한되었던한계를극복한 Rocketscript TM series 제품은 42~70 까지고객의실험조건에맞게다양한온도에서 RT 반응을수행할수있어효과적으로 cdna 를합성할수있습니다. Ease-of-use: Thermostable Reverse Transcriptase, RNase inhibitor 등 cdna 합성에필요한모든구성물질을포함하고있어증폭하고자하는 RNA 및 primer 와함께 RT 반응을수행할수있습니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Application Gene synthesis First-strand synthesis of cdna from RNA molecules (Reverse Transcription) RT-PCR Random priming reactions Library construction Probe labeling mrna 5 -end mapping by primer extension analysis Real-Time PCR Concentration 10,000 U (200 U/μl) Storage Temperature -20 Unit Definition One is defined as the amount of enzyme required to incorporates 1 nmole of dttp into acid-precipitable material in 10 min at 37 using poly A, oligo dt as template primer. www.bioneer.co.kr 311

RocketScript Reverse Transcriptase Experimental Data Target: Caspase-3 42 C 50 C 60 C A B A B A B Rocketscript TM RTase thermostable Activity 45 C 50 C 55 C 60 C 65 C 70 C 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Target: TFRC 42 C 50 C 60 C A B A B A B I Company RTase thermostable Activity 45 C 50 C 55 C 60 C 65 C 70 C 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 70 C RNA with seceondary structunes Figure 2. Thermostable stability check. Amplification results of RocketScript TM Reverse Transcriptase using myc compared with supplier I Reverse transcription. condition: Incubation at each temperature 45 /50 /55 /60 /65 /70 for 1 hr, deactivation at 95 for 5 min Primer set: human myc 495 bp set Lane 1: 100 ng human total RNA from HeLa cell Lane 2: 10 ng human total RNA from HeLa cell Lane 3: 1 ng human total RNA from HeLa cell Lane 4: 100 pg human total RNA from HeLa cell Lane M: 1 kb DNA Ladder (Bioneer, D-1040) 65 C 60 C RocketScript TM Reverse Transcriptaes TFRC Bioneer Supplier I Supplier Q Supplier A Supplier P 55 C 50 C 45 C Figure 3. Comparison of amplification quality between RocketScript TM RTase and competitor RTases. Target gene expression Level. Lane 1: 100 ng human total RNA from HeLa cell Lane 2: 10 ng human total RNA from HeLa cell Lane 3: 1 ng human total RNA from HeLa cell Lane 4: 100 pg human total RNA from HeLa cell Lane M: 1 kb DNA Ladder (Bioneer, D-1040) Figure 1. Complex RNA amplification. Complex RNA amplification results of RocketScript TM Reverse Transcriptase compared with that of conventional RTase. Reverse transcription condition: conventional 42 / 50 / 60 1 hr, deactivation 95 5 min, this product shows thermal stability. Lane A: M-MLV Reverse Transcriptase Lane B: RocketScript TM Reverse Transcriptase Lane 1: 100 ng human total RNA from HeLa cell Lane 2: 10 ng human total RNA from HeLa cell Ordering Information Product Description E-3141 RocketScript TM Reverse Transcriptase, 10,000 U (50 rxns) E-3142 RocketScript TM Reverse Transcriptase, 50,000 U (250 rxns) BIONEER 312

03 DNA Ligase T4 DNA Ligase 314 Thermostable Thermus filiformis (Tfi) DNA Ligase 315

T4 DNA Ligase For Ligation of DNA, TA Cloning, and Other Recombinant DNA Applications Reagents Supplied 10 x Reaction Buffer: 500 mm Tris-HCI (ph 7.8), 100 mm MgCl 2, 50 mm DTT, 10 mm ATP, 25 μg/ml BSA Concentration 20,000 U (200 U/μl) Storage Conditions 50% glycerol containing 10 mm Tris-HCI (ph 7.5), 50 mm KCI, 1 mm EDTA, 10 mm 2-mercaptoethanol Description 유전자재조합실험에주로사용되는본제품은 DNA 사이의결합에사용됩니다. T4 DNA ligase 는 duplex DNA 또는 RNA 에있는 5 - 인산기말단과 3 - 수산기말단부위사이에 phosphodiester bond 형성을촉진하여 duplex DNA, RNA, 또는 DNA/RNA hybrids 에있는단일가닥틈사이를 repair 할뿐만아니라 blunt-end 그리고 cohesive-end 끝말단부위로부터 DNA 를 ligate 시킵니다. Features and Benefits High Speed: cohesive end DNA 에서 25, 10 분, blunt end DNA 에서 25, 10 분반응으로 DNA ligation 이가능합니다. Flexibility: 대부분의 DNA ligation 에최적화된제품입니다. Reproducibility: 재현성있는결과를위해바이오니아의전제품은엄격한 ISO 품질시스템하에서생산됩니다. Specifications Heat Inactivation: 70 for 10 min Application Blunt or cohesive-end ligation Repair of nicks in double-stranded nucleic acids Storage Temperature -20 Unit Definition 0.01 Weiss of enzyme is defined as the amount of enzyme required to give 90% ligation of Hind III fragments of lambda DNA in 30 min, at 16 in 20 μl of the assay mixture. Experimental Data Lane 1: DNA fragment (digested with EcoR V) Lane 2, 3, 4, 5: T4 DNA Ligase 1 U, 16, 10, 20, 30 and 60 min Lane 6, 7, 8, 9: T4 DNA Ligase 1 U, 25, 10, 20, 30 and 60 min Lane 10, 11, 12, 13: T4 DNA Ligase 1 U, 37, 10, 20, 30 and 60 min Lane 14: Lambda DNA (digested with Hind III) Lane 15, 16, 17: T4 DNA Ligase 1 U, 16, 10, 20 and 30 min Lane 18, 19, 20: T4 DNA Ligase 1 U, 25, 10, 20 and 30 min Lane 21, 22, 23: T4 DNA Ligase 1 U, 37, 10, 20 and 30 min Ordering Information Product Description E-3061 T4 DNA Ligase, 20,000 U, 1 tube E-3062 T4 DNA Ligase, 100,000 U, 20,000 U x 5 tubes BIONEER 314

Thermostable Thermus filiformis (Tfi) DNA Ligase Unit definition One of Tfi DNA Ligase is defined as the amount of enzyme required to give 50% ligation of the 12 base pair cohesive ends of 1 μg of PspE I digested lambda DNA in 10 min at 45. Description Tfi DNA Ligase 는 DNA Ligase 의일종으로서절단된이중나선 DNA 분자의인접한 5 - 인산기말단과 3 - 수산기말단사이를인산디에스테르결합 (phosphodiester bond) 으로연결해주는효소입니다. 특히반응온도는 45 와 65 사이이므로다른 T4 DNA Ligase, E. coli DNA ligase 등과비교해보면더높은온도에서활성을안정하게유지하여다른 DNA Ligase 보다더높은반응온도조건에서의결합반응에사용할수있습니다. Source: Tfi DNA Ligase is isolated from E. coli cells containing the ligase gene cloned from Thermus filiformis. Application Ligase Chain Reaction (LCR) Oligonucleotide Ligation Assay (OLA) Mutagenesis by Incorporation of a phosphorylated oligo during PCR Amplification Simultaneous Mutagenesis of Multiple Sites Activity Assay Conditions The activity assay is carried out in a 20 μl reaction containing 1 μg of PspE I digested lambda DNA and 1 x Tfi DNA ligase reaction buffer. After incubation at 45 for 10 min., the reaction is terminated by addition of stop solution (40%(w/v) sucrose, 50 mm EDTA and 0.25% bromophenol blue). Then heat at 70 for 10 min and immediately load on a 0.8% agarose gel. Stability The half-life of the enzyme in 1 x reaction buffer is more than 1 hour at 95 and 55 hr at 65. Note: Tfi DNA Ligase should not be used as a substitute for other DNA ligase, i.e., T4 DNA Ligase. References Barany, F. (1991) Proc. Natl. Acad. Sci. USA, 88, 189-193. Landegren, U. et al.(1988) Science 241, 1077-1080 Michael, Scott F. (1994) Biotechniques 16:3, 410-412. Gerard J. A. et al. (1993) Biotechniques 15:1, 172-178. Reagents Supplied 10 x Reaction buffer (1 ml): 300 mm Tris-HCl (ph 8.3), 250 mm KCl, 50 mm MgCl 2, 5 mm NAD 1 x Dilution buffer (1 ml): 10 mm Tris-HCl (ph 7.6), 0.1 mm EDTA, 50 mm KCl, 1 mm DTT, 200 μg/ml acetylated BSA, 50% Glycerol Storage Condition 20 mm Tris-HCl (ph 7.6), 2 mm MgCl 2, 1 mm EDTA, 1 mm DTT, 0.5% Tween -20, 0.5% IGEPAL CA-630, 50% Glycerol, store at Concentration 20 s/μl www.bioneer.co.kr 315

Thermostable Thermus filiformis (Tfi) DNA Ligase Experimental Data 1 2 3 4 5 6 Heat Stability test at 95 C and 65 C 1 2 3 4 5 6 7 8 9 10 1. Fragment 4. Fragment Ligation Product (1+4) Figure 1. Ligation test at various temperatures (45 ~65 ) Incubate the reaction containing ligase 1 and 1 μg DNA[lambda PspE I] at each temperature for 10 min. Lane 1: λdna/pspe I (control) Lane 2: Incubate at 45, 10 min Lane 3: Incubate at 50, 10 min Lane 4: Incubate at 55, 10 min Lane 5: Incubate at 60, 10 min Lane 6: Incubate at 65, 10 min Figure 2. Heat Stability test at 95. Incubate the enzyme at 95 each time. And then add 1 ligase to a 20 μl reaction containing 1 μl DNA[lambda PspE I] and incubate the mixture at 45 for 10 min. Lane 1: λdna/pspe I (control) Lane 2: Incubate at 95, 10 min Lane 3: Incubate at 95, 20 min Lane 4: Incubate at 95, 30 min Lane 5: Incubate at 95, 40 min Lane 6: Incubate at 95, 50 min Lane 7: Incubate at 95, 60 min Lane 8: Incubate at 95, 70 min Lane 9: Incubate at 95, 80 min Lane 10: Incubate at 95, 90 min Ordering Information Product Description E-3111 Tfi DNA Ligase, 2,000 U, 10 x reaction buffer, 1 ml, 1 x dilution buffer, 1 ml E-3112 Tfi DNA Ligase, 10,000 U, 10 x reaction buffer, 1 ml, 1 x dilution buffer, 1 ml BIONEER 316

04 Restriction Enzymes Restriction Enzymes 318

Restriction Enzymes AccuCut TM Buffer Activity Chart Isoschizomer Summary List Palindromic Tetra-and Hexa-Nucleotide Recognition Sequences AfeⅠ~XmaⅠ Restriction Enzymes Bioneer provides 10X AccuCut TM buffer with every restriction endonuclease to exert optimal activity. This activity chart lists each restriction endonuclease and t s optimal AccuCut TM buffer. It also gives the approximate activity of each restriction endonuclease. All four AccuCut TM buffers are selected on the best conditions for multiple digest. Unit determination One of restriction endonuclease activity is defined the amount of enzyme required to completely digest 1 μg of substrate DNA in a total reaction volume of 50 μl in an hour using the AccuCut TM buffer provided. Incubations are performed in 1.5 ml tubes at the appropriate incubation temperature as indicated in the product profile. Digestion Assay for Nuclease Contamination All restriction endonuclease are incubated overnight in their recommended AccuCut TM buffer with 1 μg of substrate DNA in a volume of 50 μl. The characteristic banding pattern produced by the enzyme in one hour is compared to the pattern produced after overnight incubation. An unaltered pattern is an indication that the enzyme is free of detectable levels of nonspecific DNase. The maximum number of s that can be incubated overnight is given in product profile. Assay for Exonuclease and Phosphatase Contamination All restriction endonuclease are incubated with 0.1 μg of a 32P-labelled double strand oligonucleotide 25~30 bp in a 50 μl reaction volume with the supplied AccuCut TM buffer. Incubations are at the recommended temperature for 4 hr. Reaction products are separated by 20% PAGE with 7 M urea and are displayed by subsequent autoradiography of gel. Exonuclease and phosphatase contamination is determined as a percentage, calculated for 1 of the enzyme. All restriction endonuclease contain less than 0.05% of the contamination activity. Digestion-Ligation-Recutting This assay is used to test for exonuclease activity that would degrade the termini of restriction fragments, resulting in inhibition of ligation and of subsequent digestion of ligated fragments. DNA fragments are produced by an excessive over-digestion of substrate DNA with each restriction endonuclease. The fragments obtained are then ligated. The ligated fragments are then recut with the same restriction endonuclease. A normal banding pattern after cleavage indicates that both the 3 and 5 termini are intact and the enzymes preparation is free of detectable exonuclease and phosphatase. Blue-White Selection Assay Enzymes used for cloning application are tested by an Results for digestion Overdigestion assay Ligation and recutting assay Lane 1 : 1 U of BamH I Lane 2 : 1.5 U of BamH I Lane 3 : 2 U of BamH I Lane 4 : 3 U of BamH I Lane 5 : 10 U of BamH I Lane 6 : Lambda DNA Lane 1 : Lambda DNA Lane 2 : 60 U of BamH I Lane 3 : 120 U of BamH I Lane 1 : Cut Lane 2 : Ligate Lane 3 : Recut BIONEER 318

Restriction Enzymes additional quality control, the Blue-White Screening Assay, to determine the integrity of the DNA ends produced after digestion with an excess of enzyme. The assay is performed by digesting an appropriate vector at a unique site with a 5-fold excess of enzyme, ligating, transforming into JM109 cells and plating on X-gal/ ITPG/ Amp plates. The percentage of white versus colonies is then determined. Enzymes that generate overhangs must produce fewer than 2% white colonies and blunt cutting enzymes must produce fewer than 5% white colonies. All restriction enzymes tested must produce fewer than 3% white colonies in order to be Blue-White certified. 10X Reaction Buffer Bioneer provides a color coated 10X reaction buffer with each of the restriction endonuclease to ensure optimal activity. The buffer should be used at 10 times diluted concentration in the reaction. Storage All the enzymes must be stored at Buffer systems AccuCut TM buffer Blue (B) 10 mm Tris-HCl, 10 mm MgCl 2, 100 mm NaCl, 1 mm DTT, ph 8.5 AccuCut TM buffer violet (I) 33 mm Tris-acetate, 10 mm Mgacetate, 66 mm potassium-acetate, 1 mm DTT, ph 7.9 AccuCut TM buffer Orange (O) 10 mm Tris-HCl, 10 mm MgCl 2, 50 mm NaCl, 1 mm DTT, ph 7.6 AccuCut TM buffer green (N) 10 mm Tris-HCl, 10 mm MgCl 2, 1 mm DTT, ph 7.6 AccuCut TM buffer Red (R) 50 mm Tris-HCl, 10 mm MgCl 2, 100 mm NaCl, 1 mm DTT, ph 7.6 * Concentration of each enzymes is dependent on the batches of production. To know exact concentration of enzymes, please see the insert sheet in the each enzyme package. Results for digestion Overdigestion assay Ligation and recutting assay Lane 1 : Lambda DNA Lane 2 : 1 U of Ssp I Lane 3 : 2 U of Ssp I Lane 4 : 3 U of Ssp I Lane 5 : 10 U of Ssp I Lane 1 : Lambda DNA Lane 2 : 10 U of Ssp I Lane 3 : 20 U of Ssp I Lane 1 : Cut Lane 2 : Ligate Lane 3 : Recut www.bioneer.co.kr 319

Restriction Enzymes Enzyme Restriction Endonuclease Activity in the Five Standard AccuCut TM buffer Optimal Buffer Activity in AccuCut TM buffers (%) B I O N R Afe I I 75-100 100 25-50 0-25 75-100 37 Alu I N 50-75 75-100 75-100 100 0-25 37 Apa I I 0-10 100 25-50 50-75 0-10 37 AspLE I (Hha I) R 50-75 25-50 75-100 0-25 100 37 AsuNH I (Nhe I) N 0-10 75-100 50-75 100 0-10 37 BamH I O 75-100 25-50 100 25-50 75-100 37 Bgl II R 25-50 10-25 10-25 0-10 100 37 Bsa29 I (Cla I) O 50-75 75-100 100 25-50 50-75 37 Bsp19 I (Nco I) B 100 10-25 25-50 0-10 75-100 37 Bst2U I (BstN I) O 50-75 10-25 100 75-100 50-75 60 BstDE I (Dde I) O 50-75 10-25 100 75-100 25-50 60 CciN I (Not I) I 75-100 100 50-75 25-50 75-100 37 Dra I O 75-100 75-100 100 75-100 25-50 37 EcoR I EcoR I 100 50-75 75-100 50-75 75-100 37 EcoR V B 100 25-50 25-50 0-10 50-75 37 Fau I N 0-10 50-75 25-50 100 0-10 55 FauND I (Nde I) I 50-75 100 75-100 50-75 10-25 37 Hae III O 50-75 50-75 100 75-100 25-50 37 Hind III B 100 0-10 25-50 10-25 0-10 37 Hinf I R 75-100 75-100 75-100 25-50 100 37 Kpn I N 25-50 75-100 25-50 100 25-50 37 Kzo9 I (Dpn II) O 50-75 50-75 100 50-75 50-75 37 Mlu I R 25-50 10-25 10-25 0-10 100 37 Mly113 I (Nar I) N 10-25 50-75 25-50 100 10-25 37 Msp I N 75-100 75-100 75-100 100 50-75 37 Psp124B I (Sac I) O 0-10 75-100 100 75-100 10-25 37 Pst I R 25-50 25-50 25-50 10-25 100 37 Pvu II O 25-50 25-50 100 25-50 25-50 37 Rsa I N 50-75 75-100 50-75 100 0-10 37 Sal I R 25-50 0-10 10-25 0-10 100 37 SfaN I R 75-100 0-10 25-50 10-25 100 37 Sfr274 I (Xho I) N 50-75 75-100 75-100 100 50-75 50 Sma I I 0-10 100 0-10 0-10 0-10 25 Sph I (Bbu I) O 75-100 50-75 100 25-50 75-100 37 Tru9 I (Mse I) B 100 50-75 25-50 75-100 25-50 65 Vsp I (Ase I) B 100 25-50 10-25 0-10 50-75 37 Xba I R 50-75 75-100 75-100 75-100 100 37 Xma I I 0-10 100 50-75 75-100 0 37 Optimal Temp. BIONEER 320

Restriction Enzymes Restriction Enzymes Isoschizomer Contents Bioneer Enzyme Isoschizomer 인식부위 Acc16 I Aos I, Avi II, Fsp I, Mst I, Nsb I TGC GCA Acc65 I Asp718 I, Sth I G GTACC Acc113 I Dpa I, Eco255 I, Sca I AGT ACT AccB1 I Ban I, Hgic I, BbvB I, Eco64 I, MspB4 I G G(C/T)(G/A)CC AccB7 I Acp II, Asp10H II, Esp1396 I, Van91 I, PflM I CCANNNN NTGG AccBS I BsrB I, Mbi I GAG CGG Acl I Psp1406 I AA CGTT AclN I Spe I A CTAGT AclW I Bin I, Alw I, BspP I GGATCNNNN Acs I Apo I, Fsi I, Xap I (G/A) AATT(C/T) Afe I Ait I, Aor51H I, Fun I, Eco47 III AGC GCT Alu I Mlt I AG CT Ama87 I Aqu I, Ava I, Bco I, BsoB I, Eco27K I, Eco88 I, NspII, NspSA I C (C/T)CG(G/A)G Apa I Ppe I GGGCC C AsiA I Age I, PinA I, BshT I A CCGGT AspLE I Cfo I, FnuD III, Hha I GCG C AspS9 I Asu I, Avc I, BspB II, Bsu54 I, Cfr13 I, Sau96 I G GNCC AsuC2 I Aha I, Bcn I, Cau II, HgiS22 I, Nci I CC (C/G)GG AsuHP I Hph I GGTGANNNNNNNN AsuNH I Nhe I, PstNH I G CTAGC BamH I AccEB I, Ali I, Bna I, Bst I, NspSA IV, Sur I G GATCC Bbv12 I Alw21 I, AspH I, Bsh45 I, HgiA I, BsiHKA I G(A/T)GC(A/T) C Bgl I Tsp8E I GCCNNNN NGGC Bgl II Ncr I, NspMAC I, Pae2k I, Pae18k I A GATCT Bme18 I Afl I, Ava II, Bme216 I, Cau I, Eco47 I, HgiB I, HgiE I, Sin I G G(A/T)CC Bpu14 I Acp I, Asp10H I, Asu II, Bsp119 I, BstB I, Csp45 I, Fsp II, Lsp I, Nsp V, Sfu I, Ssp1 I TT CGAA Bsa29 I Aag I, Ban III, Bci29 I, Bsc I, Bsp106 I, Bsu15 I, Cla I AT CGAT Bsc4 I BsiY I, Bsl I, BsaL I CCNNNNN NNGG Bse1 I BseN I, Bsr I, BsrS I, Bst11 I, Tsp1 I ACTG G Bse3D I BsrD I, BseM I GCAATGNN Bse8 I BsaB I, Bsh1365 I, BsiB I, BsrBR I, Mam I GATNN NNATC Bse21 I Aoc I, Axy I, Bst29 I, Bst30 I, Bsu36 I, Cvn I, Eco81 I, Mst II, Sau I, SshA I CC TNAGG Bse118 I Bco118, BsrF I, BssA I, Cfr10 I (G/A) CCGG(C/T) BseP I BssH II, Pau I G CGCGC BseX3 I Aaa I, BstZ I, Eag I, EclX I, Eco52 I, Xma III C GGCCG Bsp13 I Acc III, BseA I, BsiM I, BspE I, BspM II, Bsu23 I, Kpn2 I, Mro I, Pta I T CCGGA Bsp19 I Nco I C CATGG Bsp1720 I Blp I, Bpu1102 I, Cel II, Esp I GC TNAGC www.bioneer.co.kr 321

Restriction Enzymes BIONEER BspA2 I Avr II, AvrB II, Bln I C CTAGG BssNA I BspM90 I, BstBS I, Bst1107 I, Sna I, Xca I GTA TAC BssT1 I EcoT14 I, Eco130 I, Erh I, ErhB9 II, Sty I C C(A/T)(A/T)GG Bst2B I Bsi I, BssS I C TCGTG Bst2U I Aor I, Apy I, BseB I, Bse16 I, Bse17 I, Bse24 I, BspN I, Bst2 I, BstN I, BstO I, EcoR II, Fsp1604 I, Mva I, Sth117 I, Zan I CC (A/T)GG BstAC I Acy I, Aha I, Asu III, Bbi II, BsaH I, Hgi I, Hin1 I, Msp17 I, Pam II G(G/A) CG(C/T)C BstAP I ApaB I GCANNNN NTGC BstBA I BsaA I, MspY I (C/T)AC GT(G/A) BstDE I Dde I C TNAG BstDS I Dsa I C C(A/G)(C/T)GG BstF5 I BseG I GGATGNN BstH2 I AccB2 I, Bsp143 II, Hae II (G/A)GCGC (C/T) BstHP I Hpa I GTT AAC BstMC I BsaO I, Bsh1285 I, BsiE I, Mcr I CG(G/A)(C/T) CG BstNS I Nsp I, NspH I (G/A)CATG (C/T) BstSF I Sfc I, Sfe I C T(G/A)(C/T)AG BstSN I Eco105 I, SnaB I TAC GTA BstX2 I BstY I, Mfl I, Tru201 I, Xho II (G/A) GATC(C/T) Bsu6 I Bco5 I, Bco116 I, BseZ I, Ear I, Ksp632 I CTCTTCN BsuR I Bim19 II, Bsh I, BspK I, BspR I, Bsp211 I, Dsa II, FnuD I, Hae III, Pla I, Sbv I, Sfa I GG CC CciN I Not I GC GGCCGC Dra I Aha III TTT AAA DseD I Drd I GACNNNN NNGTC EcoR I Hal I, Kpn49k I, Rsr I, Sso I G AATTC EcoR V Ceq I, Eco32 I GAT ATC Ege I Ehe I, Eco78 I GGC GCC Erh I BssT1 I, EcoT14 I, Eco130 I, ErhB9 II, Sty I C C(A/T)(A/T)GG Fau I None CCCGCNNNN FauND I Nde I CA TATG Fok I None GGATG(9) FriO I Ban II, Bsu1854 I, Bsp519 I, Bvu I, Eco24 I, Eco215 I, HgiJ II, SacN I G(G/A)GC(C/T) C Fsp4H I BsoF I, Bsp6 I, Fbr I, Fnu4H I, Ita I, Uur960 I GC NGC Hae III Bim19 II, Bsh I, BspK I, Bsp211 I, BsuR I, Dsa II, FnuD I, Pla I, Sbv I, Sfa I GG CC Hind III BstF I, EcoV III, Hsu I, Ssb I A AGCTT Hinf I CviB I, FnuA I, Hha II G ANTC Hpa II Bco27 I, BsiS I, Bst40 I, Hap II, Msp I, Sth134 I C CGG HspA I HinP1 I, Hin6 I, SciN I G CGC Kpn I None GGTAC C Ksp22 I AtuC I, Bco102 I, BspX II, Fba I, Pov I T GATCA Kzo9 I AspMD I, BspA I, Bsp105 I, Bsp143 I, BtK II, Dpn II, Mbo I, Nde II, Nla II, Sau3A I GATC 322

Restriction Enzymes Mlu I None A CGCGT Mly113 I Mch I, Nar I, Nda I, Nun II, SseA I GG CGCC MroN I Eco56 I, NgoA IV, NgoM IV G CCGGC MroX I Asp700 I, BbuA I, Xmn I GAANN NNTTC Msp I Hpa II와동일 C CGG MspR9 I Bme1390 I, Msp67 I, ScrF I CC NGG Nru I Bsp68 I, MluB2 I, Sbo13 I, Spo I TCG CGA NruG I Ahd I, AspE I, Eam1105 I, EclHK I, Uba1190 I, Uba1191 I GACNNN NNGTC Ple19 I BspC I, ErhB9 I, Nb1 I, Ple19 I, Pvu I, Rsh I, Xor II CGAT CG Pme55 I Aat I, Eco147 I, SseB I, Stu I AGG CCT Psp124B I Sac I, Sst I GAGCT C PspE I Acr II, AspA I, BstE II, BstP I, Eca I, EcoO65 I, Eco91 I, NspSA II G GTNACC PspL I BpuB5 I, BsiW I, Pfl23 II, PpuA I, Sp1 I, Sun I C GTACG PspN4 I AspN I, BscB I, Nla IV GGN NCC PspOM I Bsp120 I G GGCCC PspPP I Pfl27 I, PpuM I, Psp5 II (G /A) GG(A/T)CC(C/T) Pst I Api I, Asp713 I, BspB I, Bsp63 I, Hal II, Sfl I CTGCA G Pvu II Bav I, Dma I, Pvu84 II CAG CTG Rsa I Afa I GT AC Sal I HgiC III, HgiD II, Nop I, Xci I G TCGAC Sbf I Sse8387 I CCTGCA GG SfaN I None GCATCNNNNN Sfi I Sdi I GGCCNNNN NGGCC Sfr274 I Abr I, Blu I, Ccr I, Mav I, PaeR7 I, Pan I, Sla I, Xho I, Xpa I C TCGAG Sfr303 I Cfr42 I, Csc I, Gal I, Kpn19 I, Ksp I, Sac II, Sst II CCGC GG Sma I CfrJ4 I, PaeB I, PspAL I CCC GGG Smi I Swa I ATTT AAAT Sph I Bbu I, Pae I GCATG C Sse9 I TspE I, Tsp509 I AATT Ssp I unfound AAT ATT Tru9 I Mse I, Tru1 I T TAA Tth111 I Asp I, Ats I GACN NNGTC Vha464 I Afl II, Bfr I, BspT I, Bst98 I, Esp4 I, MspC I C TTAAG Vne I Aaq I, Alw44 I, ApaL I, Sno I G TGCAC Vsp I Ase I, Asn I, PshB I, Vsp I AT TAAT Xba I None T CTAGA Xma I Ahy I, Cfr9 I, EaeA I, PspA I, Xcy I, XmaC I C CCGGG Zsp2 I EcoT22 I, Mph1103 I, Nsi I, PinB I, Sep I ATGCA T Ksp22 I AtuC I, Bco102 I, BspX II, Fba I, Pov I T GATCA Kzo9 I AspMD I, BspA I, Bsp105 I, Bsp143 I, BtK II, Dpn II, Mbo I, Nde II, Nla II, Sau3A I GATC www.bioneer.co.kr 323

Restriction Enzymes Palindromic Tetra-and Hexa-Nucleotide Recognition Sequences AATT ACGT AGCT ATAT CATG CCGG CGCG CTAG GATC GCGC GGCC GTAC TATA TCGA TGCA TTAA Ssep I TspE I Kzo9 I Bsp143 I Mbo I Mae Ⅱ Hpa Ⅱ Msp I Mae I Hsp I Hin6 I Csp6 I Taq I Tru9 I Tru1 I Alu I Bsh1236 I Dpn I BsuR I Hae Ⅲ Rsa I AspLE I Hha I Tai I Nla Ⅲ A T Apo I Hind Ⅲ Bsp- LU11 I AsiA I Age I BsaW I Cfr10 I Mlu I Afl I AclN I Spe I Bgi I Xho Ⅱ Pru10 I A T Acl I Rsp1406 I Bsa29 I Bsu15 I Vap I A T Sap I Afe I Eco47 I Pme55 I Eco147 I Acc13 I Eco255 I Sca I A T BIONEER 324

Restriction Enzymes AATT ACGT AGCT ATAT CATG CCGG CGCG CTAG GATC GCGC GGCC GTAC TATA TCGA TGCA TTAA A T Nsp I Bsp142 Ⅱ Zsp2 I Mph103 I C G Bsp19 I BseD I Bsa I Eco130 I Nco I Xma I BseD I Cfr9 I Eco88 I BseD I Dsa I BspA2X I Avr I BseD I Eco130 I BseX3 I Cfr I Eco52 I Sfr274 I PspL I Pfl23Ⅱ Bfm I Eco88 I Xho I Bfm I Vha464 I BspT I C G FauND I Nde I C G Eco72 I BsaA I Pvu Ⅱ NapB Ⅱ Sme I NspB Ⅱ C G Sfr303 I Cfr42 I Ple19 I Bsh1258 I Pvu I Bsh1258 I C G Pst I G C EcoR I Api I MroN I NgoM Ⅳ BseP I Pau I AsuNH I Nhe I BamH I Xho Ⅱ BshN I Eco64 I Kas I PspCM I Bsp120 I Acc651 I BshN I Eco64 I Sal I Vne I Alw441 I G C Ecl136 Ⅱ Mly113 Hin1 I Nar I Acc Acc I G C Ecl136 Ⅱ EcoR V Eco32 I Cac8 I Nae I Cac8 I Cac8 I Cac8 I BspL I Ege I Ehe I BspL I BspL I BspL I BssNA I Bst1107 I HinC Ⅱ BstHP I HinC Ⅱ Hpa I G C G C Aat I PspLE I Alw21 I Eco24 I Sac I Sdu I Sph I Pae I Nsp I Bsp143 Ⅱ Bbe I Apa I Eco24 I Sdu I Kpn I Alw21 I Sdu I T A BspH I Bsp13 I BsaW I Kpn2 I Xba I Bcl I Cfr I Ksp22 I Bsp1407 I T A Bpu14 I Bsp119 I T A BstSN I Eco105 I BsaA I Nru I Bsp68 I Acc16 I Fsp I Bal I Dra I T A T A www.bioneer.co.kr 325