CAT. NO. BR BR BR SIZE 8 reactions 40 reactions 80 reactions. Reagent BISULFITE 2 ml 2 2 ml 3 2 ml

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1 Product Insert LOT: See product label EXPIRY DATE: See product label ORDERING INFORMATION PRODUCT CAT. NO. BR BR BR SIZE 8 reactions 40 reactions 80 reactions COMPONENTS Reagent BISULFITE 2 ml 2 2 ml 3 2 ml Buffer CONVERSION 2 ml 2 ml 2 2 ml Buffer BINDING BS (concentrate) 7 ml (add 7 ml ethanol) 30 ml (add 30 ml ethanol) 30 ml (add 30 ml ethanol) Buffer WASH WB (concentrate) 2 ml (add 18 ml ethanol) 10 ml (add 90 ml ethanol) 10 ml (add 90 ml ethanol) Buffer DESULFONATION (concentrate) 3 ml (add 9 ml ethanol) 7.5 ml (add 22.5 ml ethanol) 15 ml (add 45 ml ethanol) Buffer ELUTION 2 ml 3 2 ml 15 ml Mini Filters (orange) Collection Tubes (2.0 ml) Elution Tubes (1.5 ml) STORAGE Room temperature (until expiry date see product label). If precipitation appears, gently warm the solution to dissolve the precipitate. After opening, the kit components can be used for up to one month. FEATURES Fast and easy bisulfite conversion with high conversion rates Flexible use with a wide range of sample types Including subsequent desulfonation and DNA cleanup APPLICATIONS Bisulfite conversion of unmethylated cytosines to uracils for epigenomics studies PIN page 1

2 DESCRIPTION biotechrabbit GenUP Bisulfite Kit provides a fast and easy method for the conversion of unmethylated cytosines to uracils enabling DNA methylation studies (see Fig. 1 and Fig. 2). The kit is designed for high conversion rates, extremely easy application and exceptionally fast, reliable results. Denaturation and bisulfite treatment are performed in a single tube in approximately 3 h. After conversion, the DNA is cleaned up and desulfonated on a Mini Filter and can be used for downstream applications, such as PCR and sequencing. The GenUP Bisulfite Kit can be used directly with purified DNA. It contains all reagents and consumables for convenient bisulfite conversion. When starting from tissues, cells or other DNA sources, the GenUP Bisulfite Complete Kit (BR ) offers bisulfite conversion in combination with genomic DNA extraction. Otherwise, use one of biotechrabbits GenUP Kits for purification of genomic DNA prior to bisulfite conversion. Fig. 1. Conversion of cytosine to uracil. While unmethylated cytosine is deamidated to uracil during the reaction, 5-methylcytosine resists bisulfite treatment. AATC m GCATC m Bisulfit Conversion GTCA Fig. 2. Effect of bisulfite treatment on DNA sequence. AATC m GUATC m GTUA SPECIFICATIONS STARTING MATERIAL PREPARATION TIME Purified DNA (500 pg to 10 µg) Conversion reaction: 45 min Purification and desulfonation: 45 min MATERIALS SUPPLIED BY THE USER 70% ethanol % ethanol 0.2 ml, 1.5 ml or 2.0 ml reaction tubes Pipet tips page 2 info@biotechrabbit.com PIN

3 STEPS BEFORE STARTING Add the following volume of % ethanol to each bottle, close firmly, mix thoroughly and store at room temperature. CAT. NO. CONCENTRATE ETHANOL FINAL VOLUME Buffer BINDING BS Buffer WASH WB Buffer DESULFONATION BR ml 7 ml 14 ml BR ml 30 ml 60 ml BR ml 30 ml 60 ml BR ml 18 ml 20 ml BR ml 90 ml 100 ml BR ml 90 ml 100 ml BR ml 9 ml 12 ml BR ml 22.5 ml 30 ml BR ml 45 ml 60 ml All centrifugation steps should be carried out at room temperature. Avoid repeated freezing and thawing of starting material. Before use, ensure all kit components are at room temperature. Set thermal mixer, water bath or optional PCR cycler to 85 C prior to bisulfite conversion. Mark all vials and filters to avoid confusion when purifying multiple preps. IMPORTANT NOTES Once opened Reagent BISULFITE can be stored for 1 month at room temperature. Improper storage of the Reagent BISULFITE can lead to its degradation. In the first place this degradation can be caused by atmospheric oxygen. Degradation can be detected by ph decrease (below ph 5.3), loss of viscosity, and lightly lucid yellow color. If in doubt, the ph value should be tested with ph-paper (not provided in the kit). Do not use the Reagent BISULFITE, if ph is lower than 5.1. Reagent BISULFITE and Buffer CONVERSION tend to form two phases. For successful bisulfite conversion mix bisulfite reaction mix vigorously and spin down briefly in order to remove drops from the lid. Otherwise these droplets will not reach the proper temperature and the conversion will not be complete. For the bisulfite reaction only use reaction tubes which fit properly into the respective device (thermal mixer with 2 ml or 1.5 ml tube racks, thermal cycler with 200 µl tube rack) in order to ensure an optimal heat exchange at 85 C. We recommend using a shaking platform (thermal mixer, water bath or another rocking platform) for a continuous shaking of the sample. Alternatively, vortex the sample 3 5 times during incubation. No shaking will reduce the conversion reaction efficiency. The incubation conditions must be maintained stringently. The samples have to be cleaned up and desulfonated immediately after bisulfite conversion! PIN page 3

4 SHORT PROTOCOL STEPS SCHEME Preparation of bisulfite reaction mixture. Bisulfite conversion reaction for 45 min at 85 C. Sample cleanup by binding of DNA to Mini Filter (orange). Desulfonate and wash the bound DNA. Elution of converted DNA. page 4 info@biotechrabbit.com PIN

5 PROTOCOL FOR BISULFITE CONVERSION PROCEDURE Transfer 500 pg to 10 µg DNA (in 50 µl) to a 0.2 ml, 1.5 ml or 2.0 ml reaction tube. Add 70 µl Reagent BISULFITE and 30 µl Buffer CONVERSION. Mix vigorously by pulse vortexing for 10 s. Centrifuge briefly to remove the drops from the lid. Incubate at 85 C for 45 min with shaking at 800 rpm. Centrifuge briefly and continue immediately with the next step. NOTES Be sure to use reaction tubes that fit properly into the heating device to ensure optimal heat exchange. The Reagent BISULFITE can degrade when stored improperly. If the ph of the Reagent BISULFITE is less than 5.1, do not use. Be sure the Reagent BISULFITE and Buffer CONVERSION are mixed thoroughly. Adhere to incubation conditions stringently. Ensure the incubation temperature is maintained constantly at 85 C. Use a shaking platform (thermomixer, water bath or other rocking platform) to ensure continuous shaking during lysis. Alternatively, vortex the sample 3 5 times during the incubation. Failure to shake samples reduces the efficiency of the conversion reaction. Samples must be cleaned up and desulfonated immediately after conversion. Transfer the sample to a 1.5 ml or 2.0 ml reaction tube if the sample is in a tube of a different size. Add 700 µl Buffer BINDING BS and mix by vortexing or by pipetting up and down. Ensure the Buffer BINDING BS has been prepared as described above. It is important that the sample is mixed to homogeneity. Transfer the sample to a Mini Filter (orange) placed in a Collection Tube. Centrifuge at 14,000 g (16,000 rpm) for 3 min. Discard the Collection Tube with the filtrate. Place the Mini Filter into a new Collection Tube. Add 200 µl Buffer WASH WB. Centrifuge at 14,000 g (16,000 rpm) for 1 min. Discard the Collection Tube with the filtrate. Place the Mini Filter into a new Collection Tube. Add 700 µl Buffer DESULFONATION. Incubate at room temperature for 10 min. Centrifuge at 14,000 g (16,000 rpm) for 1 min. Discard the Collection Tube with the filtrate. If the solution does not pass completely through the Mini Filter, repeat the centrifugation or use a longer centrifugation time. Ensure the Buffer WASH WB has been prepared as described above. Ensure the Buffer DESULFONATION has been prepared as described above. Place the Mini Filter into a new Collection Tube. Add 400 µl Buffer WASH WB. Centrifuge at 14,000 g (16,000 rpm) for 1 min. Discard the filtrate and re-use the Collection Tube. PIN page 5

6 Place the Mini Filter into the same Collection Tube. Add 400 µl Buffer WASH WB. Centrifuge at 14,000 g (16,000 rpm) for 1 min. Discard Collection Tube with the filtrate. Transfer the Mini Filter to a new Collection Tube. Centrifuge at 14,000 g (16,000 rpm) for 3 min to remove all traces of ethanol. Discard Collection Tube with the filtrate. Transfer the Mini Filter to an Elution Tube. Incubate the sample at 60 C for 10 min with the lid open. Add 50 µl Buffer ELUTION. Incubate at room temperature for 1 min. Centrifuge at 8000 g (10,000 rpm) for 1 min. Do not transfer Buffer WASH WB to the Elution Tube. To improve yield, perform elution twice using ½ volume of Buffer ELUTION. Purified DNA in the Elution Tube can be used immediately. Store the DNA at 4 C (short-term) or 20 C (long-term). page 6 info@biotechrabbit.com PIN

7 TROUBLESHOOTING PROBLEM SOLUTION LOW YIELD Incomplete elution Insufficient mixing with Buffer BINDING BS Reagent BISULFITE decomposed Increase the elution time up to 5 min, or repeat the elution. Use a higher elution volume or elute in two steps. Mix the sample with Buffer BINDING BS by pipetting or vortexing before transferring to the Mini Filter. Use a fresh aliquot of Reagent BISULFITE. LOW CONCENTRATION DN A Excessive Buffer ELUTION Elute the converted DNA in a smaller volume of Buffer ELUTION (minimum 20 µl) POOR PERFORMANCE IN PCR AMPLIFICATION OF DNA ISOLATED FROM FFPE TISSUE Inappropriate PCR conditions Increase polymerase concentration in the PCR. Prolong elongation time during PCR. Design shorter PCR amplicons. SAFETY PRECAUTIONS This kit is made for single use only! Don t eat or drink components of the kit! The kit shall only be handled by educated personal in a laboratory environment! Wear gloves while handling these reagents, and avoid skin contact! In case of contact, flush with water immediately! Handle and discard waste according to local safety regulations! Do not add bleach or acidic components to the waste after sample preparation! Buffer BINDING BS contains guanidine isothiocyanate, which is harmful to health. Contact with acids liberates very toxic gas! PIN page 7

8 CERTIFICATE OF ANALYSIS The components of the kit were tested by real-time PCR and melting curve analysis in a comparison of treated and untreated samples using components from different production lots. Initially a sequencing analysis of treated and untreated DNA samples was performed to confirm bisulfite conversion efficiency. Purified DNA has been positively tested. Quality confirmed by: Head of Quality Control SAFETY INSTRUCTIONS For safety instructions please see Safety Data Sheets (SDS)/Sicherheitshinweise finden Sie in den SDS unter: USEFUL HINTS Visit Applications at for more products and product selection guides. Most biotechrabbit products are available in custom formulations and bulk amounts. CONTACT BIOTECHRABBIT biotechrabbit GmbH Volmerstr. 9a Berlin, Germany info@biotechrabbit.com support@biotechrabbit.com Phone: Fax: Legal Disclaimer and Product Use Limitation Purchase of product does not include a license to perform any patented applications; therefore it is the sole responsibility of users to determine whether they may be required to engage a license agreement depending upon the particular application in which the product is used. This product was developed, manufactured, and sold for in vitro use only. It is not suitable for administration to humans or animals. Trademarks: biotechrabbit, GenUP (biotechrabbit GmbH). valid from page 8 info@biotechrabbit.com PIN