GenUP Bisulfite Complete Kit

Transcription

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 Buffer LYSIS LK 2 ml 10 ml 2 10 ml Proteinase K 2 vials (add 0.3 ml water) 1 vial (add 1.5 ml water) 2 vials (add 1.5 ml water) Reagent BISULFITE 2 ml 2 2 ml 3 2 ml Buffer CONVERSION 2 ml 2 ml 2 2 ml Buffer BINDING BS 7 ml (add 7 ml ethanol) 30 ml (add 30 ml ethanol) 30 ml (add 30 ml ethanol) Buffer WASH WB 2 ml (add 18 ml ethanol) 10 ml (add 90 ml ethanol) 10 ml (add 90 ml ethanol) Buffer DESULFONATION 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. Store lyophilized Proteinase K at 4 C, Store aliquots of dissolved Proteinase K at 20 C. 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 cells, tissues and FFPE samples Including subsequent desulfonation and DNA cleanup Isolation of both modified and unmodified genomic DNA in parallel PIN page 1

2 APPLICATIONS Isolation of genomic DNA with subsequent bisulfite conversion of unmethylated cytosines to uracils in a wide range of sample types and subsequent desulfonation and DNA cleanup. DESCRIPTION biotechrabbit GenUP Bisulfite Kit provides a fast and easy method for the isolation of genomic DNA from various sources in combination with 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. Both modified and unmodified genomic DNA can be isolated in parallel in one experiment setup. 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 purified DNA can then be stored at 20 C or used for subsequent applications. 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 EXTRACTION TIME Purified DNA (1 pg to 10 µg) Cells (maximum ) Fresh tissue (maximum 1 mg) FFPE tissue sections (1 3 sections of maximum 10 µm each) FFPE tissue punch biopsy core (maximum 10 mg) Bronchial aspirates, swabs, peritoneal cavity fluid, pleural effusions, sputum, urine sediment Lysis: approximately 20 min to 48 hours, depending on the sample type Conversion reaction: 45 min Purification and desulfonation: 45 min page 2 info@biotechrabbit.com PIN

3 MATERIALS SUPPLIED BY THE USER 70% and % ethanol 0.2 ml, 1.5 ml or 2.0 ml reaction tubes Pipet tips Double-distilled water Dithiothreitol (DTT) Phosphate buffered saline (PBS) GenUP FFPE Paraffin Removal_Solution (BR ) STEPS BEFORE STARTING PURIFICATION Add the following volume of % ethanol to the 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 Add the following volume of double-distilled water to each vial Proteinase K, mix thoroughly and store aliquots at 20 C. BR BR , BR ml 1.5 ml for ml aliquots Avoid repeated freezing and thawing of starting material. All centrifugation steps should be carried out at room temperature. Before use, ensure all kit components are at room temperature. Set thermal mixer or water bath to 60 C if sample lysis is required. 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. PIN page 3

4 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! page 4 info@biotechrabbit.com PIN

5 SHORT PROTOCOL STEPS SCHEME Lysis of starting material (if required). 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. PIN page 5

6 PROTOCOL FOR SAMPLE PREPARATION: PURIFIED DNA PROCEDURE NOTES Transfer 500 pg to 10 µg (in 50 µl) to a 0.2 ml, 1.5 ml or 2.0 ml reaction tube. Continue with bisulfite conversion on page 9. PROTOCOL FOR SAMPLE PREPARATION: CULTURED CELLS PROCEDURE Transfer up to cells to a 2.0 ml reaction tube. Centrifuge at 3000 x g (5000 rpm) for 5 min to pellet cells. Discard the supernatant. NOTES Smaller numbers of cells can be processed by adjusting reagent volumes accordingly. For mucin-rich samples: bring the total volume to 1.8 ml PBS. Add 20 µl 0.5 mg/ml DTT and mix vigorously by vortexing. Incubate at room temperature for 30 min. Centrifuge at 3000 x g (5000 rpm) for 5 min. Discard supernatant. Repeat this step if necessary. For samples preserved with cross-linking or precipitating fixatives (ethanol, Saccomanno s fixative, formalin, etc.): wash twice with PBS. Add 190 µl Buffer LYSIS LK and 15 µl Proteinase K and mix by pulse vortexing for 10 sec. Centrifuge briefly to remove drops from the lid. Incubate at 60 C for 3 h with shaking at 800 rpm. Samples fixed with cross-linking fixatives should be incubated up to 24 h. In this case, add additional 15 µl Proteinase K after 8 16 h. For samples fixed with cross-linking fixatives: incubate at 95 C for 30 min after lysis. Before use, prepare Proteinase K as described above. Use a shaking platform (thermomixer, water bath or other rocking platform) to ensure continuous shaking during lysis. Alternatively, vortex the sample 3 4 times during the incubation. Centrifuge briefly. Transfer 50 µl lysate to a 0.2 ml, 1.5 ml or 2.0 ml reaction tube. Continue with bisulfite conversion on page 9. Optionally: Transfer 150 µl residual lysate for direct isolation of native genomic DNA in a separate tube (1.5 ml or 2.0 ml). Continue with sample cleanup on page 10. page 6 info@biotechrabbit.com PIN

7 PROTOCOL FOR SAMPLE PREPARATION: FRESH TISSUE PROCEDURE Transfer up to 1 mg fresh tissue to a 2.0 ml reaction tube. Homogenize the sample. Add 190 µl Buffer LYSIS LK and 15 µl Proteinase K and mix vigorously by vortexing for 10 sec. Centrifuge briefly to remove drops from the lid. NOTES Smaller amounts of fresh tissue can be processed by adjusting reagent volumes accordingly. Before use, prepare Proteinase K as described above. Incubate at 60 C for 3 h with shaking at 800 rpm. Use a shaking platform (thermomixer, water bath or other rocking platform) to ensure continuous shaking during lysis. Alternatively, vortex the sample 3 4 times during the incubation. Centrifuge briefly. Transfer 50 µl lysate to a 0.2 ml, 1.5 ml or 2.0 ml reaction tube. Continue with bisulfite conversion on page 9 Optionally: Transfer 150 µl residual lysate for direct isolation of native genomic DNA in a separate tube (1.5 ml or 2.0 ml). Continue with sample cleanup on page 10. PROTOCOL FOR SAMPLE PREPARATION: FFPE TISSUE SECTIONS PROCEDURE Transfer 1 3 sections (maximum 10 µm each) of FFPE tissue to a 2.0 ml reaction tube. Centrifuge at maximum speed for 1 min. Add 190 µl Buffer LYSIS LK and 15 µl Proteinase K and mix by pulse vortexing for 10 sec. Centrifuge briefly to remove drops from the lid. Incubate at 60 C for 3 h with shaking at 800 rpm. For improved lysis, the incubation can be extended up to 48 h. In this case, add an additional 15 µl Proteinase K after 8 16 h. Incubate at 95 C for 30 min. Centrifuge briefly. Transfer 50 µl lysate to a 0.2 ml, 1.5 ml or 2.0 ml reaction tube. Continue with bisulfite conversion on page 9. NOTES Deparaffinization is not necessary. Larger or smaller amounts of FFPE tissue can be processed by adjusting reagent volumes accordingly. Before use, prepare Proteinase K as described above. Ensure that the tissue is covered with liquid. Use a shaking platform (thermomixer, water bath or other rocking platform) to ensure continuous shaking during lysis. Alternatively, vortex the sample 3 4 times during the incubation. This step removes remaining cross-links. Optionally: Transfer 150 µl residual lysate for direct isolation of native genomic DNA in a separate tube (1.5 ml or 2.0 ml). Continue with sample cleanup on page 10. PIN page 7

8 PROTOCOL FOR SAMPLE PREPARATION: FFPE TISSUE PUNCH BIOPSY CORES PROCEDURE To remove the paraffin from the FFPE tissue biopsy cores, place the tissue in a 2.0 ml reaction tube and add 3 5 drops of GenUP FFPE Paraffin Removal_Solution (not provided). Add 190 µl Buffer LYSIS LK and 15 µl Proteinase K and mix by pulse vortexing for 10 sec. NOTES Smaller amounts of FFPE tissue punch biopsy cores can be processed by adjusting reagent volumes accordingly. The FFPE slices dissolve immediately. Add enough solution to submerge the FFPE slices (approximately µl). See the product insert of GenUP FFPE Paraffin Removal_Solution (BR ) for more detailed information. Before use, prepare Proteinase K as described above. For better lysis, break up the tissue with a pipette tip. Centrifuge briefly to remove drops from the lid. Ensure that the tissue is covered with liquid. Incubate at 60 C up to 48 h with shaking at 800 rpm. After 24 h, add 15 µl Proteinase K Use a shaking platform (thermomixer, water bath or other rocking platform) to ensure continuous shaking during lysis. Alternatively, vortex the sample 3 4 times during the incubation. Incubate at 90 C for 10 min. This step removes remaining cross-links. Centrifuge briefly to separate phases. The upper, orange phase contains the paraffin. Transfer 50 µl of the lower colorless aqueous phase containing the genomic DNA to a 0.2 ml, 1.5 ml or 2.0 ml reaction tube. Continue with bisulfite conversion, page 9. Optionally: Transfer 150 µl residual lysate for direct isolation of native genomic DNA in a separate tube (1.5 ml or 2.0 ml). Continue with sample cleanup on page 10. page 8 info@biotechrabbit.com PIN

9 PROTOCOL FOR BISULFITE CONVERSION PROCEDURE To the prepared sample (50 µl), add 70 µl Reagent BISULFITE and 30 µl Buffer CONVERSION. Mix vigorously by pulse vortexing for 10 sec. Centrifuge briefly to remove the drops from the lid. NOTES Use DNA (500 pg 100 µg DNA) or crude lysate prepared as described above. 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. Incubate at 85 C for 45 min with shaking at 800 rpm. 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 4 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. Centrifuge briefly. Transfer the complete sample (150 µl) to a 1.5 ml or 2.0 ml reaction tube in case the bisulfite conversion was performed in a different reaction tube. Continue immediately with desulfonation and sample clean-up, page 10. PIN page 9

10 PROTOCOL FOR DESULFONATION AND SAMPLE CLEAN-UP Add 700 µl Buffer BINDING BS to the sample and mix by vortexing or by pipetting up and down. 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. Ensure the Buffer BINDING BS has been prepared as described above. It is important that the sample is mixed to homogeneity. 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. 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. Transfer the Mini Filter to an Elution Tube. Dry the sample at 60 C for 10 min with the lid open in a thermal mixer. 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 10 info@biotechrabbit.com PIN

11 TROUBLESHOOTING PROBLEM SOLUTION CLOGGED MINI FILTER Too much starting material or insufficient lysis Reduce the amount of starting material. Increase the lysis time. Increase the centrifugation speed. After lysis, centrifuge the lysate to pellet unlysed material. Increase the volume of Buffer LYSIS LK and Proteinase K. LOW YIELD Insufficient lysis Incomplete elution Insufficient mixing with Buffer BINDING BS Reagent BISULFITE decomposed Increase lysis time. Reduce the amount of starting material. Do not overload the Mini Filter 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 Buffer REAGENT. 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 Insufficient lysis Inappropriate PCR conditions Increase lysis time. Increase Proteinase K concentration. 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 11

12 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. The following sample types have been positively tested: purified DNA samples, FFPE tissue sections, FFPE tissue punch biopsy cores, cells, bronchial aspirates, swabs, peritoneal cavity fluid, pleural effusions, sputum, urine sediment and fresh tissue. 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 12 info@biotechrabbit.com PIN