QPCR-S100 (100 rxns) Store -20. Functional analysis RealHelix qpcr Kit was evaluated by real-time PCR using the 10-fold serial-diluted human

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1 RealHelix TM qpcr Kit [Intercalator type] CERTIFICATE OF ANALYSIS (1603-V01R03) Kit contents RealHelix TM qpcr Kit [Intercalator type] Cat. No. QPCR-S100 (100 rxns) QPCR-S500 (500 rxns) 2x qpcr PreMix [S] 1.25 ml x 2ea 1.25 ml x 10ea ROX Passive Reference Dye 0.1 ml 0.1 ml x 5ea Certificate Analysis 1 ea 1 ea Description The RealHelix qpcr Kit is designed to perform a rapid real-time quantification of target DNA or 1 st -strand cdna synthesized from RNA molecule. This kit includes a double strand DNA specific-binding fluorescent dye showing several-fold higher fluorescence than SYBR Green and little inhibition to the PCR process. The kit consists of convenient 2x concentrated premix solution and ROX passive dye. The 2x premix contains hot-start PCR enzyme, dntps, buffers, Mg 2+ and stabilizing agent. The hot-start PCR enzyme provides high specific amplification of target DNA and minimizes the side products such as primer dimers. Included ROX dye could be used as a passive reference dye. Application Quantification of target DNA sample by real time PCR Store -20 2x qpcr PreMix and ROX Passive Reference Dye should be stored in a dark. Quality control assay data Functional analysis RealHelix qpcr Kit was evaluated by real-time PCR using the 10-fold serial-diluted human genomic DNA and a set of human gene-specific primer with ABI 7900HT Fast-real-time PCR system (Applied Biosystems, USA) and Rotor-Gene Q instrument (QIAGEN, Germany). Quality authorized by Youn Taek Go

2 qpcr Kit [Intercalator type] Protocol 1. Program a real-time PCR instrument according to the recommendations below. The excitation and emission maxima of the included SYBR Green I are at 494 nm and 521 nm, respectively. <2-step cycling protocol> If annealing temperature(at) of primers used in real-time PCR is between 58 and 62, thermo cycling can be performed using 2-step cycling protocol as follow. Step Condition Cycle(s) Enzyme activation 95 for 12 ~ 15 min 1 PCR Amplification Denaturation Annealing & extension 95 for 20 sec ª60 for 40 sec Collect the fluorescence data 40 For melting curve analysis of products, refer to the instrument documentation <3-step cycling protocol> If annealing temperature(at) of primers used in real-time PCR is under 58 or above 62, thermo cycling can be performed using 3-step cycling protocol as follow. Step Condition Cycle(s) Enzyme activation 95 for 12 ~ 15 min 1 Denaturation 95 for 20 sec PCR Amplification Annealing Extention ªAT for 30 sec 72 for 30 sec Collect the fluorescence data 40 For melting curve analysis of products, refer to the instrument documentation

3 qpcr Kit [Intercalator type] 2. Add following components for a single 50 μl reaction volume.* Components Volumes DNA Template (1 ng ~ 1 μg) 2x qpcr PreMix Forward primer (10 pmoles/μl) Reverse primer (10 pmoles/μl) ROX Passive Reference Dye (Optional) DEPC-treated water X μl 25 μl 2 μl 2 μl 1 or 0.1 μl Adjust to final 50 μl * For multiple reactions, prepare a master mix by adding the required volumes of each above components (except the DNA template) and dispense appropriate volumes into PCR tubes or plates. * The reaction volume for a reaction could be adjusted according to the recommendations for the instruments. See the following table for amount or concentration of ROX Passive Reference Dye to use for your specific instrument Instruments Amount of ROX per 50 μl Rxn Final ROX Conc. ABI 7000, 7300, 7700, 7900HT, 7900HT Fast, StepOne, StepOnePlus ABI 7500, 7500 Fast. Stratagene Mx3000, Mx3005P, Mx μl 500 nm 0.1 μl 50 nm Bio-Rad icycler iq, iq5 and MyiQ, Opticon, CFX 96, CFX 384 Roche LightCycler 480, LightCycler 2.0 Qiagen Rotor-Gene Q Eppendorf MasterCycler ep realplex Cepheid Smart Cycler Not required Not required To accurately pipet 0.1 μl per reaction, use 1 μl of 10 fold diluted ROX dye. The diluted ROX dye should be prepared immediately before use. 3. Gently mix and immediately centrifuge the reaction mix. 4. Perform the Real-time PCR.

4 qpcr Kit [Intercalator type] Products Cat. No. Products Size QPCR-S100 RealHelix qpcr Kit [Intercalator type] 100 rxns QPCR-S500 RealHelix qpcr Kit [Intercalator type] 500 rxns

5 CERTIFICATE OF ANALYSIS (1603-V01R03) RealHelix TM qpcr Kit [Probe type] Kit contents RealHelix TM qpcr Kit [Probe type] Cat. No. QPCR-P100 (100 rxns) QPCR-P500 (500 rxns) 2x qpcr PreMix [P] 1.25 ml x 2ea 1.25 ml x 10ea ROX Passive Reference Dye 0.1 ml 0.1 ml x 5ea Certificate Analysis 1 ea 1 ea Description The RealHelix qpcr Kit [Probe type] is designed to perform a rapid real-time quantification of target DNA or 1 st -strand cdna using dual-labeled probe. The kit consists of convenient 2x concentrated premix solution and ROX passive dye. The 2x premix contains hot-start PCR enzyme, dntps, buffers, Mg 2+ and stabilizing agent. The hot-start PCR enzyme provides high specific amplification of target DNA and minimizes the side products such as primer dimers. Included ROX dye could be used as a passive reference dye. Application Quantification of target DNA sample by real time PCR Store -20 ROX Passive Reference Dye should be stored in a dark. Quality control assay data Functional analysis RealHelix qpcr Kit was evaluated by real-time PCR using the 10-fold serial-diluted human genomic DNA and a set of human gene-specific primer and dual-labeled probe with ABI 7900HT Fast-real-time PCR system (Applied Biosystems, USA) and Rotor-Gene Q instrument (QIAGEN, Germany). Quality authorized by Youn Taek Go

6 qpcr Kit [Probe type] Protocol 1. Program a real-time PCR instrument according to the recommendations below. <2-step cycling protocol> If annealing temperature(at) of primers used in real-time PCR is between 58 and 62, thermo cycling can be performed using 2-step cycling protocol as follow. Step Condition Cycle(s) Enzyme activation 95 for 12 ~ 15 min 1 PCR Amplification Denaturation Annealing & extension 95 for 20 sec ª60 for 40 sec Collect the fluorescence data 40 <3-step cycling protocol> If annealing temperature(at) of primers used in real-time PCR is under 58 or above 62, thermo cycling can be performed using 3-step cycling protocol as follow. Step Condition Cycle(s) Enzyme activation 95 for 12 ~ 15 min 1 Denaturation 95 for 20 sec PCR Amplification Annealing Extention ªAT for 30 sec 72 for 30 sec Collect the fluorescence data 40 Guideline for designing a dual labeled probe - Check that the probe sequence contains more C residues than G residues - Do not put a G on 5 -end. - The optimal probe T m should be ~ 10 higher than primers Set-up the excitation and emission wavelength of instrument according to the fluorescent dye of dual-labeled probe used in experiment. For melting curve analysis of products, refer to the instrument documentation

7 qpcr Kit [Probe type] 2. Add following components for a single 50 μl reaction volume.* Components Volumes DNA Template (1 ng ~ 1 μg) 2x qpcr PreMix [P] Forward primer (10 pmoles/μl) Reverse primer (10 pmoles/μl) Probe (5 pmoles/μl) ROX Passive Reference Dye (Optional) DEPC-treated water X μl 25 μl 2 μl 2 μl 1 μl 1 or 0.1 μl Adjust to final 50 μl * For multiple reactions, prepare a master mix by adding the required volumes of each above components (except the DNA template) and dispense appropriate volumes into PCR tubes or plates. * The reaction volume for a reaction could be adjusted according to the recommendations for the instruments. See the following table for amount or concentration of ROX Passive Reference Dye to use for your specific instrument. Instruments Amount of ROX per 50 μl Rxn Final ROX Conc. ABI 7000, 7300, 7700, 7900HT, 7900HT Fast, StepOne, StepOnePlus ABI 7500, 7500 Fast. Stratagene Mx3000, Mx3005P, Mx μl 500 nm 0.1 μl 50 nm Bio-Rad icycler iq, iq5 and MyiQ, Opticon, CFX 96, CFX 384 Roche LightCycler 480, LightCycler 2.0 Qiagen Rotor-Gene Q Eppendorf MasterCycler ep realplex Cepheid Smart Cycler Not required Not required To accurately pipet 0.1 μl per reaction, use 1 μl of 10 fold diluted ROX dye. The diluted ROX dye should be prepared immediately before use. 3. Gently mix and immediately centrifuge the reaction mix. 4. Perform the Real-time PCR.

8 qpcr Kit [Probe type] Products Cat. No. Products Size QPCR-P100 RealHelix qpcr Kit [Probe type] 100 rxns QPCR-P500 RealHelix qpcr Kit [Probe type] 500 rxns