instructions for use - analytik jena ag · 9 general procedure for dna extraction and bisulfite ......

Instructions for UseLife Science Kits & Assays

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innuCONVERT Bisulfite All-In-One Kit

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Order No.:845-IC-2000008 8 reactions845-IC-2000040 40 reactions845-IC-2000080 80 reactions

Publication No.: HB_IC-2000_e_170518

This documentation describes the state at the time of publishing.It needs not necessarily agree with future versions. Subject to change!

Print-out and further use permitted with indication of source.© Copyright 2017, Analytik Jena AG, AJ Innuscreen GmbH

Manufacturer:AJ Innuscreen GmbHRobert-Rössle-Straße 1013125 BerlinMade in Germany!

Distribution/Publisher:Analytik Jena AGKonrad-Zuse-Straße 107745 Jena · Germany

Phone +49 3641 77 9400Fax +49 3641 77 [email protected]

Contents

innuCONVERT Bisulfite All-In-One Kit Issue 05 / 2017 1

Contents

1 Introduction ........................................................................................... 3

1.1 Intended use ................................................................................ 3

1.2 Notes on the use of this manual ................................................. 5

2 Safety precautions ................................................................................. 6

3 Storage conditions ................................................................................ 8

4 Functional testing and technical assistance ......................................... 8

5 Product use and warranty ..................................................................... 9

6 Kit components .................................................................................. 10

7 GHS classification ............................................................................... 13

7.1 Hazard phrases ......................................................................... 14

7.2 Precaution phrases ................................................................... 15

8 Recommended steps before starting ................................................ 16

9 General procedure for DNA extraction and bisulfite conversion ...... 17

10 Product specifications ........................................................................ 18

11 Lysis Protocols: Sample preparation/DNA isolation prior to bisulfite conversion .......................................................................................... 19

11.1 Protocol 1: Purified DNA .......................................................... 19

11.2 Protocol 2: Formalin-fixed and paraffin-embedded (FFPE) tissue sections .......................................................................... 19

11.3 Protocol 3: FFPE tissue punch biopsy cores ............................ 21

11.4 Protocol 4: Cell lines/cultures and cytological samples .......... 23

11.5 Protocol 5: Fresh tissue ............................................................ 25

12 Preparation of bisulfite reaction mixture .......................................... 26

13 Bisulfite conversion reaction ............................................................. 28

14 Desulfonation reaction and sample clean-up ................................... 29

14.1 Protocol A: high purity of eluted DNA ..................................... 30

14.2 Protocol B: fast clean-up and reagent saving ......................... 32

Contents

2 Issue 05 / 2017 innuCONVERT Bisulfite All-In-One Kit

15 Troubleshooting ................................................................................. 34

16 Related Products ................................................................................ 35

Introduction


1 Introduction

1.1 Intended use

DNA methylation is an epigenetic mechanism which is found in most or-ganisms. Its main functions in prokaryotes refer to the protection against parasitic DNA and replication fidelity control. In vertebrates the predomi-nant methylation site is the 5´-carbon of cytosine within the CpG dinu-cleotide context. More than half of the genes contain short (~ 1 kb) CpG-rich regions known as CpG islands while the rest of the genome is deplet-ed for CpGs. While CpG sites within CpG islands are mainly unmethylated, most CpG sites outside CpG islands are usually methylated. The function of CpG methylation has proved to be challenging to unravel and seems to vary with context. CpG methylation is involved in several fundamental mechanisms, i.e. gene regulation, X-chromosome inactivation, genomic imprinting and maintenance of chromosomal stability [for review: Jones PA, 2012. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat Rev Genet. 13(7):484-92.].

Furthermore, aberrant DNA methylation is a hallmark of malignant tu-mors and reflects an early event during carcinogenesis. In tumors the global genome methylation decreases (genome-wide hypomethylation), while several specific genes are methylated (gene specific hypermethyla-tion). Hypomethylation typically affects repetitive DNA elements, while hypermethylation usually occurs within CpG islands.

Nowadays the DNA methylation analysis has become an important tool for studying cancer, gene expression, genetic diseases and many other important aspects of biology. The testing of DNA methylation has also become more and more relevant in medical diagnostics.

Most techniques for studying DNA methylation depend on a preceding deamination of cytosines by means of bisulfite. In the presence of bisul-fite unmethylated cytosines are converted to uracils, while methylated cytosines remain unaffected. Accordingly, the epigenetic information is converted into DNA sequence information and therefore can be deter-mined by standard molecular biological methods such as PCR and DNA sequencing.

Introduction


CONSULT INSTRUCTION FOR USEThis package insert must be read carefully prior to use. Package insert instructions must be followed accordingly. Reliability of results cannot be guaranteed if there are any deviations from the instructions in this pack-age insert.

The innuCONVERT Bisulfite All-In-One Kit includes all necessary reagents and consumables for the conversion of unmethylated cytosines to uracils (Figure 1 and 2). After conversion, the DNA must be cleaned up and desulfonated by the included reagents. The purified DNA can then be stored at -22 °C to -18 °C or used for subsequent applications.

GCmGTAGCAACmG GCGTAGCAACG

Bisulfite Conversion

GCmGTAGUAACmG GUGTAGUAAUG

Figure. 2: Effect of bisulfite treatment on DNA sequence. Conversion of epigenetic information into sequence information.

Figure 1: Conversion of unmethylated cytosine to uracil. Cytosines are deaminated to uracils in the presence of bisulfite while methylated cy-tosines remain unaffected.

Introduction


1.2 Notes on the use of this manual

For easy reference and orientation, the manual uses the following warn-ing and information symbols as well as the shown methodology:

Symbol Information

REF Catalogue number.

N Content Contains sufficient reagents for <N> reactions.

Storage conditions Store at room temperature or shown conditions respectively.

Consult instructions for use This information must be observed to avoid improper use of the kit and the kit components.

Expiry date

Lot number The number of the kit charge.

Manufactured by Contact information of manufacturer.

For single use only Do not use components for a second time.

Note / Attention Observe the notes marked in this way to ensure correct function of the device and to avoid operating errors for obtaining correct re-sults.

The following systematic approach is introduced in the manual:

The chapters and figures are numbered consecutively.

A cross reference is indicated with an arrow (e.g. "Notes on the use of this manual" p. 5).

Working steps are numbered.

30 °C

15 °C

Safety precautions


2 Safety precautions

NOTE Read through this chapter carefully prior to guarantee your own safety and a trouble-free operation. Follow all the safety instructions explained in the manual, as well as all messages and information, which are shown.

All due care and attention should be exercised in handling the materials and reagents contained in the kit. Always wear gloves while handling these reagents and avoid any skin contact! In case of contact, flush eyes or skin with a large amount of water immediately.

FOR SINGLE USE ONLY! This kit is made for single use only!

ATTENTION! Don’t eat or drink components of the kit! The kit shall only be handled by educated personnel in a laboratory envi-ronment!

If the buffer bottles are damaged or leaking, wear gloves and protective googles when discarding the bottles in order to avoid any injuries. Analyt-ik Jena AG has not tested the liquid waste generated during using the kit for potential residual infectious components. This case is highly unlikely, but cannot be excluded completely. Therefore, all liquid waste must be considered as potentially infectious and must be handled and discarded according to local safety regulation.

Safety precautions


Clinical sample must always be considered as potentially infectious. Sam-ples from risk patients must always be labeled and handled under conse-quent safety conditions. Please observe the federal, state and local safety and environmental regulations. Follow the usual precautions for applica-tions using extracted nucleic acids. All materials and reagents used for DNA or RNA isolation should be free of DNases or RNases.

ATTENTION! Do not add bleach or acidic components to the waste after sample prepa-ration!

NOTE Emergency medical information in English and German can be obtained 24 hours a day from: Poison Information Center, Freiburg / Germany Phone: +49 (0)761 19 240.

For more information, please ask for the material safety data sheet (MSDS).

Storage conditions


3 Storage conditions

The innuCONVERT Bisulfite All-In-One Kit should be stored dry, dark at room temperature (15 °C to 30 °C). When stored at these conditions, the kit is stable until the expiration date printed on the label on the kit box.

For further information see chapter ”Kit components“ p. 10.

NOTE After opening of the individual components the kit is stable for at least 1 month!

4 Functional testing and technical assistance

The Analytik Jena AG guarantees the correct function of the kit for appli-cations as described in the manual. This product has been produced and tested in an ISO 13485 certified facility.

We reserve the right to change or modify our products to enhance their performance and design. If you have any questions or problems regarding any aspects of the innuCONVERT Bisulfite All-In-One Kit or other Analytik Jena AG products, please do not hesitate to contact us. For technical sup-port or further information in Germany please dial +49 36 41 / 77 94 00. For other countries please contact your local distributor.

Product use and warranty


5 Product use and warranty

The kit is not designed for the usage of other starting materials or other amounts of starting materials than those, referred to in the manual ( “Intended use” p. 3) ( “Product specifications” p. 18). Since the per-formance characteristics of Analytik Jena AG kits have just been validated for the application described above, the user is responsible for the valida-tion of the performance of Analytik Jena AG kits using other protocols than those described below. Analytik Jena AG kits may be used in clinical diagnostic laboratory systems after the laboratory has validated the com-plete diagnostic system as required by CLIA’ 88 regulations in the U.S. or equivalent regulations required in other countries.

All products sold by the Analytik Jena AG are subjected to extensive quali-ty control procedures and are warranted to perform as described when used correctly. Any problems should be reported immediately.

NOTE For research use only!

Kit components


6 Kit components

IMPORTANT Store lyophilized Proteinase K at 4 °C to 8 °C! Divide dissolved Proteinase K into aliquots and storage at -22 °C to -18 °C is recommended. Repeated freezing and thawing will reduce the activity dramatically!

STORAGE CONDITIONS All other components are stored at room temperature. Protect the rea-gents from exposure to direct light.

8 40 80

845-IC-2000008 845-IC-2000040 845-IC-2000080

Lysis Solution BC 2 ml 10 ml 20 ml

Proteinase K For 1 x 0.3 ml working solution

For 1 x 1.5 ml working solution

For 1 x 1.5 ml working solution

Conversion Reagent 2 ml 2 x 2 ml 3 x 2 ml Conversion Buffer 2 ml 2 ml 2 x 2 ml Binding Solution GS (conc.)

5 ml

15 ml

30 ml

Washing Solution BS (conc.)

1 ml

5 ml

10 ml

Washing Solution C 5 ml 25 ml 50 ml Desulfonation Buffer (conc.)

2 x 2 ml 5 x 2 ml 10 x 2 ml

Elution Buffer 2 ml 2 x 2 ml 3 x 2 ml Spin Filter 8 40 2 x 40 Receiver Tubes 60 6 x 50 12 x 50 Elution Tubes 8 40 2 x 40 Manual 1 1 1

30 °C

15 °C

Kit components


8 40 80

845-IC-2000008 845-IC-2000040 845-IC-2000080

Initial steps Binding Solution GS (conc.) Add 5 ml of 96-99.8 % ethanol to each bottle and mix thoroughly. Keep the bottle al-ways firmly closed!

Washing Solution BS (conc.) Add 9 ml of 96-99.8 % ethanol to each bottle and mix thoroughly. Keep the bottle al-ways firmly closed!

Proteinase K Dissolve by addi-tion of 0.3 ml of ddH2O, mix thor-oughly and store as described below!

Binding Solution GS (conc.) Add 15 ml of 96-99.8 % ethanol to each bottle and mix thoroughly. Keep the bottle al-ways firmly closed!



Binding Solution GS (conc.) Add 30 ml of 96-99.8 % ethanol to each bottle and mix thoroughly. Keep the bottle al-ways firmly closed!



Desulfonation Buffer (conc.) Prepare a multiplication of 175 μl of Desulfonation Buffer (con-centrate) and 525 μl of 96-99.8 % ethanol per every planned bi-sulfite conversion reaction shortly prior to the experiment. Pref-erably, the final mixture volume includes 10 % excess for volume loss from pipetting, i.e. for 10 reactions mix 1.925 ml of Desul-fonation Buffer (concentrate) with 5.775 ml of ethanol. Mix gen-tly by inverting the tube several times. Do not store ready-to-use Desulfonation Buffer. It is not recommended to store Desulfona-tion Buffer (concentrate) after the first opening.

Kit components


Components not included in the kit

0.2 ml, 1.5 ml, or 2.0 ml reaction tubes (preferably safe-lock) for conversion reaction

Polypropylene (PP) tubes for preparation of ready-to-use Desulfona-tion Buffer

70 % and 96–99.8 % ethanol

ddH2O for dissolving Proteinase K

Dithiothreitol (DTT),

Phosphate Buffered Saline (PBS)

Xylene, Limonene

GHS classification


7 GHS classification

Component Hazard contents GHS Symbol Hazard phrases

Precaution phrases

Binding Solu-tion GS (conc.)

Guanidinium thiocyanate 50-100 % Acetic acid 1-<2.5 % Polyethylene glycol octylphenol ether 0.3-<1.0 %

Danger

302+312+332, 314, 412

101, 102, 103, 260, 303+361+353, 305+351+338, 310, 405, 501

Conversion Buffer

1,4-Dihydroxy-benzene ≤10%, 1-Methoxy-2-propanol 50-100 %

Danger

226, 317, 318, 336, 341, 351, 400, 410

101, 102, 103, 210, 303+361+353, 305+351+338, 310, 405, 501

Conversion Reagent

Ammonium hydro-gensulphite 50-100 %

Warning

319 101, 102, 103, 280, 305+351+338

Desulfonati-on Buffer (conc.)

Sodium hydroxide 3-<5 %

Danger

314, 318 101, 102, 103, 260, 310, 305+351+338,303+361+353, 405, 501

Proteinase K Proteinase, engyo-dontium album

Danger

315, 319, 334, 317, 335

101, 102, 103, 261, 280, 305+351+338, 342+311, 405, 501

GHS classification


CAUTION DO NOT add bleach or acidic solutions directly to the sample-preparation waste.

7.1 Hazard phrases

226 Flammable liquid and vapor.

302 Harmful if swallowed.

312 Harmful in contact with skin.

314 Causes severe skin burns and eye damage.

315 Causes skin irritation.

317 May cause an allergic skin reaction.

318 Causes serious eye damage.

319 Causes serious eye irritation.

332 Harmful if inhaled.

334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.

335 May cause respiratory irritation.

336 May cause drowsiness or dizziness.

341 Suspected of causing genetic defects.

351 Suspected of causing cancer.

400 Very toxic to aquatic life.

410 Very toxic to aquatic life with long lasting effects.

412 Harmful to aquatic life with long lasting effects.

GHS classification


7.2 Precaution phrases

101 If medical advice is needed, have product container or label at hand.

102 Keep out of reach of children.

103 Read label before use.

210 Keep away from heat, hot surfaces, sparks, open flames and other ignition sources. No smoking.

260 Do not breathe dust/fume/gas/mist/vapors/spray.

261 Avoid breathing dust/fume/gas/mist/vapors/spray.

280 Wear protective gloves/protective clothing/ eye protec-tion/face protection.

310 Immediately call a POISON CENTER/doctor.

405 Store locked up.

501 Dispose of contents/container in accordance with lo-cal/regional/national/international regulations.

303+361+ 353

IF ON SKIN (or hair): Take off immediately all contami-nated clothing. Rinse skin with water/shower.

305+351+ 338

IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.

342+311 If experiencing respiratory symptoms: Call a POISON CENTER/doctor.

Recommended steps before starting


8 Recommended steps before starting

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 reaction.

Ensure that the Binding Solution GS, Washing Solution BS, Desul-fonation Buffer and Proteinase K have been prepared according to the instruction ( "Kit components" p. 10).

Centrifugation steps should be carried out at room temperature.

Avoid freezing and thawing of starting material.

General procedure for DNA extraction and bisulfite conversion


9 General procedure for DNA extraction and bisulfite conversion

Lysis of starting material (if is required) Preparation of bisulfite reaction mixture and bisulfite con-version reaction

Binding of DNA onto Spin Filter Desulfonation and washing of the bound DNA Elution of converted DNA

Product specifications


10 Product specifications

1. Starting material:

Purified DNA samples (5 ng–5 μg)

FFPE tissue sections (1–3 sections, max. 10 μm each section)

FFPE tissue punch biopsy cores (10 mg)

Cell lines (max. 5 x 105 cells)

Bronchial aspirates, swabs, peritoneal cavity fluid, pleural effu-sions, sputum, urine sediment

Fresh tissue (max. 1 mg)

2. Time for isolation:

Lysis: Approximately 30 minutes to 48 hours (depending on the sample type)

Conversion reaction: 45 minutes

Purification and desulfonation: 35 minutes

3. Positive tested for:

Purified DNA samples

FFPE tissue sections

FFPE tissue punch biopsy cores

Cell lines

Bronchial aspirates, swabs, peritoneal cavity fluid, pleural effu-sions, sputum, urine sediment

Fresh tissue

Lysis Protocols: Sample preparation/DNA isolation prior to bisulfite conversion


11 Lysis Protocols: Sample preparation/DNA isolation prior to bisulfite conversion

IMPORTANT Ensure that the Binding Solution GS, Washing Solution BS, Desulfona-tion Buffer and Proteinase K have been prepared according to the in-struction ( "Kit components" p. 10).

11.1 Protocol 1: Purified DNA

NOTE No sample preparation is necessary!

11.2 Protocol 2: Formalin-fixed and paraffin-embedded (FFPE) tissue sections

IMPORTANT Use only 1–3 FFPE sections (max. 10 μm)!

1. Provide up to three FFPE tissue sections of max. 10 μm in a 2.0 ml reaction tube. Centrifuge the reaction tube at maximum speed for 1 minute.

2. Open the reaction tube and add 190 μl Lysis Solution BC and 15 μl of Proteinase K to the sample. Mix vigorously by pulsed vortexing for 10 seconds.

NOTE Spin down briefly to remove drops from the lid. Make sure that the whole tissue section is covered with liquid.

Incubate at 60 °C for 3 hours in a thermal mixer under continuous shaking at 800 rpm.



NOTE We recommend using a shaking platform (thermal mixer, water bath or another rocking platform) for continuous shaking of the sample. Alterna-tively, vortex the sample 3–4 times during incubation. No shaking will re-duce the lysis efficiency.

3. Incubate the sample for 30 minutes at 95 °C in a thermal mixer or in a water bath in order to remove remaining crosslinks.

NOTE Deparaffinization is not necessary! DNA recovery from FFPE tissue sections depends on optimal lysis. To achieve a better lysis the incubation at 60 °C can be extended up to 48 hours. In this case an additional aliquot of 15 μl Proteinase K solution should be added after 8–16 hours. By processing higher amounts/sizes of FFPE tissue sections the volumes of reagents should be increased accordingly. The residual lysate can be cleaned up with the same procedure as the bi-sulfite treated DNA: Add 700 μl Binding Solution GS to the 150 μl of residual lysate and pro-ceed the sample purification parallel to the bisulfite treated DNA accord-ing to the instructions ("Desulfonation reaction and sample clean-up" p. 29, step 2). If small samples are prepared reduce the volume of Lysis Solution BC and Proteinase K accordingly in order to allow for an input of the whole sam-ple into the bisulfite reaction.



11.3 Protocol 3: FFPE tissue punch biopsy cores

IMPORTANT Use only 10 mg of FFPE tissue punch biopsy cores!

Deparaffinization:

1. Add 1 ml xylene or limonene (not provided in the kit) to the FFPE tissue punch biopsy cores provided in 2.0 ml reaction tubes. Incubate the reaction tube at 60 °C for 30 minutes.

2. Centrifuge at 15,000 x g (17,000 rpm) for 2 minutes.

3. Decant xylene or limonene and repeat the treatment once.

4. Add 1 ml of 96–99.8 % ethanol and incubate at room temperature for 5 minutes.

5. Centrifuge at 15,000 x g (17,000 rpm) for 2 minutes and decant the ethanol carefully.

6. Add 1 ml of 70 % ethanol and incubate at room temperature for 5 minutes.

7. Centrifuge at 15,000 x g (17,000 rpm) for 2 minutes and discard the ethanol carefully.

8. Dry the deparaffinated tissue with open caps at 60 °C until ethanol is evaporated completely.

Lysis:

1. Add 190 μl Lysis Solution BC and 15 μl of Proteinase K to the sam-ple. Mix vigorously by pulsed vortexing for 10 seconds.

NOTE Spin down briefly to remove drops from the lid. Make sure that the whole tissue section is covered with liquid.



2. Incubate at 60 °C for 48 hours in a thermal mixer under continuous shaking at 800 rpm.

NOTE We recommend using a shaking platform (thermal mixer, water bath or another rocking platform) for a continuous shaking of the sample. Alter-natively, vortex the sample 3–4 times during incubation. No shaking will reduce the lysis efficiency.

3. Incubate the sample for 30 minutes at 95 °C in a thermal mixer or in a water bath in order to remove remaining crosslinks.

NOTE After 24 hours an additional aliquot of 15 μl Proteinase K solution should be added. For better lysis the FFPE tissue punch biopsy cores can be disintegrated with a pipette tip. The residual lysate can be cleaned up with the same procedure as the bi-sulfite treated DNA: Add 700 μl Binding Solution GS to the 150 μl of residual lysate and pro-ceed the sample purification parallel to the bisulfite treated DNA accord-ing to the instructions ("Desulfonation reaction and sample clean-up" p. 29, step 2). If small samples are prepared then reduce the volume of Lysis Solution BC and Proteinase K accordingly in order to allow for an input of the whole sample into the bisulfite reaction.



11.4 Protocol 4: Cell lines/cultures and cytological samples

IMPORTANT Use max. 5 x 105 cells! Cells from cell cultures or other cellular samples, i.e. cytological samples (cellular fractions of bronchial aspirates, swabs, peritoneal cavity fluid, pleural effusions, sputum, urine sediment), should be pelleted prior to processing. Therefore, centrifuge the sample at 3,000 x g for 5 minutes and discard the supernatant. Mucin-rich samples should be pretreated with DTT (not provided in kit) in order to reduce mucin. Therefore, fill up the sample with an appropriate volume of PBS (not provided in kit) to reach a total of 1.8 ml of sample volume. Add 20 μl DTT solution (0.5 g/l) and mix vigorously by vortexing. Incubate the sample at room temperature for 30 minutes. Centrifuge the sample at 3,000 x g for 5 minutes and discard the supernatant. Repeat this procedure if necessary. Cytological samples preserved with cross-linking or precipitating fixatives (ethanol, Saccomanno’s fixative, formalin, etc.) should be washed twice with PBS (not provided in kit) prior to lysis.

1. Add 190 μl Lysis Solution BC and 15 μl of Proteinase K to the sam-ple. Mix vigorously by pulsed vortexing for 10 seconds.

NOTE Spin down briefly to remove drops from the cap.





Samples fixed with precipitating or cross-linking fixatives should incubate up to 24 hours. In this case an additional aliquot of 15 μl Proteinase K so-lution should be added after 8–16 hours. Samples fixed with cross-linking fixatives should be incubated for 30 minutes at 95 °C after lysis. The residual lysate can be cleaned up with the same procedure as the bi-sulfite treated DNA: Add 700 μl Binding Solution GS to the 150 μl of residual lysate and pro-ceed the sample purification parallel to the bisulfite treated DNA accord-ing to the instructions ("Desulfonation reaction and sample clean-up" p. 29, step 2). If small samples are prepared then reduce the volume of Lysis Solution BC and Proteinase K accordingly in order to allow for an input of the whole sample into the bisulfite reaction.



11.5 Protocol 5: Fresh tissue

IMPORTANT Use no more than 1 mg fresh tissue! Homogenize fresh tissues with a suitable tool as for example Speed Mill PLUS with corresponding Lysis Tubes (Analytik Jena AG).

1. Open the reaction tube and add 190 μl Lysis Solution BC and 15 μl of Proteinase K to the sample. Mix vigorously by pulsed vortexing for 10 seconds.

NOTE Spin down briefly to remove drops from the lid.



The residual lysate can be cleaned up with the same procedure as the bi-sulfite treated DNA: Add 700 μl Binding Solution GS to the 150 μl of residual lysate and pro-ceed the sample purification parallel to the bisulfite treated DNA accord-ing to the instructions ("Desulfonation reaction and sample clean-up" p. 29, step 2). If small samples are prepared then reduce the volume of Lysis Solution BC and Proteinase K accordingly in order to allow for an input of the whole sample into the bisulfite reaction.

Preparation of bisulfite reaction mixture


12 Preparation of bisulfite reaction mixture

IMPORTANT Once opened the Conversion Reagent can be stored 1 month. Improper storage of Conversion Reagent 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 5.1), loss of vis-cosity, 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 Conversion Reagent, if pH is lower than 5.1. Conversion Reagent may form crystals on the outskirts of the unopened vials, what does not influence the com-ponent performance. Avoid the carryover of these crystals into the bisul-fite reaction mixture. If more crystals appear in the solution after the first opening of the vial, do not use this vial again. Conversion Buffer is highly photosensitive. The prolonged contact with light may result in the colour change from lightly yellow to dark brown. However, the Conversion Buffer colour change does not influence the Kit performance. To avoid the colour change, the exposure of Conversion Buffer to light should be minimized. Conversion Reagent and Conversion Buffer tend to form two phases. For successful bisulfite conversion mix bisulfite reaction mix vigorously.

1. Prepare the bisulfite reaction mix as describe in the following table:

Reagents Amount / reaction

DNA (5 ng–5 μg) or crude lysate 50 μl

Conversion Reagent 70 μl

Conversion Buffer 30 μl

Final volume 150 μl

2. Mix vigorously by pulsed vortexing. Spin down briefly in order to re-move drops from the lid.

Preparation of bisulfite reaction mixture


NOTE 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.

Bisulfite conversion reaction


13 Bisulfite conversion reaction

IMPORTANT The bisulfite reaction mixture needs to be mixed properly prior to the incubation in order to ensure a complete conversion. The incubation conditions must be maintained stringently. It is essential to stop the incubation after 45 minutes. Prolongation of the exposure to the Conversion Reagent causes serious problems with further methylation analysis. The samples have to be cleaned up and desulfonated immedi-ately after the conversion! Be careful that no droplets remain in the reaction tube cap. These drop-lets will not reach the proper temperature and the conversion will not be complete. In order to avoid this, shortly centrifuge the reaction tube with the ready reaction mix prior to incubation. It is important to maintain the incubation temperature of 85 °C. If a ther-mal mixer is used, it should be controlled regularly. The usage of a water bath or PCR cycler is preferential.

1. Incubate the reaction mixture at 85 °C for 45 minutes in heating block, PCR cycler, water bath or thermal mixer under continuous shaking at 800 rpm.

NOTE We recommend using a shaking platform (thermal mixer, water bath or another rocking platform) for a continuous shaking of the sample. Alter-natively, vortex the sample 3–4 times during incubation. No shaking will reduce the conversion reaction efficiency.

Desulfonation reaction and sample clean-up


14 Desulfonation reaction and sample clean-up

IMPORTANT Set the thermal mixer to 60 °C! Ensure that the Binding Solution GS and Washing Solution BS have been prepared according to the instruction ( "Kit components" p. 10). Prepare the ready-to-use Desulfonation Buffer in polypropylene (PP) tubes (not supplied with the Kit) shortly prior to the experiment in the multiples for the number of the bisulfite conversion reactions to be done. For this purpose, mix gently 175 μl of Desulfonation Buffer (concentrate) with 525 μl of ethanol per each bisulfite conversion reaction. Preferably, the fi-nal mixture volume includes 10 % excess for volume loss from pipetting, i.e. for 10 reactions mix 1.925 ml of Desulfonation Buffer (concentrate) with 5.775 ml of ethanol. Do not store ready-to-use Desulfonation Buff-er. Desulfonation Buffer may form precipitates, which does not influence on the Kit’s performance. Avoid the transfer of precipitates into the Spin Filter. Do not try to dissolve precipitates by warming up.

Selecting optimal clean-up method

After the desulfonation step, two methods of DNA clean up are possible:

if your DNA will be used for downstream applications, which require ultra clean starting material (i.e. sequencing, SybrGreen® PCR), fol-low the Protocol A,

if your downstream application is not very demanding, but you wish to save your time and chemicals (ethanol absolute), follow the Pro-tocol B.

It is recommended to follow both protocols with aliquots of the same sample when using this Kit for the first time and to make a comparison of target downstream application results.



14.1 Protocol A: high purity of eluted DNA

1. After completion of the conversion reaction, briefly spin down the reaction tube and transfer the sample into a 1.5 ml or 2.0 ml reac-tion tube. No transfer is needed if the reaction was already carried out in a 1.5 ml or 2.0 ml reaction tube.

2. Add 700 μl Binding Solution GS to the converted sample, mix by vortexing or by pipetting up and down several times.

NOTE It is important that the sample and the Binding Solution GS are mixed vigorously to get a homogeneous solution.

3. Apply the sample to the Spin Filter located in a Receiver Tube. Close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute.

NOTE If the solution has not completely passed through the Spin Filter, centri-fuge again at higher speed or prolong the centrifugation time.

Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.

4. Open the Spin Filter and add 200 μl Washing Solution BS, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Dis-card the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.

5. Open the Spin Filter and add 700 μl ready-to-use Desulfonation Buffer, close the cap and incubate at room temperature for 10 minutes. Centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.



6. Open the Spin Filter and add 500 μl Washing Solution C, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Dis-card the filtrate and place the Spin Filter into a new Receiver Tube.

7. Open the Spin Filter and add 650 μl Washing Solution BS, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Dis-card the filtrate and place the Spin Filter into a new Receiver Tube.

8. Open the Spin Filter and add 650 μl ethanol absolute, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.

9. Open the Spin Filter and add 650 μl ethanol absolute, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate.

10. Place the Spin Filter into an Elution Tube.

NOTE Avoid the carryover of ethanol residuals into the Elution Tubes.

Carefully open the cap of the Spin Filter and dry the Spin Filter by incubation at 60 °C for 10 minutes in a thermal mixer.

11. Add 50 μl Elution Buffer to the Spin Filter. Incubate at room temper-ature for 1 minute. Centrifuge at 8,000 x g (10,000 rpm) for 1 minute. Two elution steps with equal volumes of Elution Buffer (e.g. 25 μl + 25 μl) might increase the yield of converted DNA.

NOTE The converted DNA can be eluted with a lower or a higher volume of Elu-tion Buffer (depends on the expected yield of converted DNA). Elution with lower volumes of Elution Buffer increases the final concentration of converted DNA. Store the converted DNA at 4–8 °C. For long time storage placing at -22 °C to -18 °C is recommended.



14.2 Protocol B: fast clean-up and reagent saving

1. After completion of the conversion reaction, briefly spin down the reaction tube and transfer the sample into a 1.5 ml or 2.0 ml reac-tion tube. No transfer is needed if the reaction was already carried out in a 1.5 ml or 2.0 ml reaction tube.

2. Add 700 μl Binding Solution GS to the converted sample, mix by vortexing or by pipetting up and down several times.

NOTE It is important that the sample and the Binding Solution GS are mixed vigorously to get a homogeneous solution.

3. Apply the sample to the Spin Filter located in a Receiver Tube. Close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute.

NOTE If the solution has not completely passed through the Spin Filter, centri-fuge again at higher speed or prolong the centrifugation time.

Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.

4. Open the Spin Filter and add 200 μl Washing Solution BS, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Dis-card the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.

5. Open the Spin Filter and add 700 μl ready-to-use Desulfonation Buffer, close the cap and incubate at room temperature for 10 minutes. Centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Discard the Receiver Tube with the filtrate. Place the Spin Filter into a new Receiver Tube.



6. Open the Spin Filter and add 400 μl Washing Solution BS, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Dis-card the filtrate and place the Spin Filter into a new Receiver Tube.

7. Open the Spin Filter and add 400 μl Washing Solution BS, close the cap and centrifuge at 14,000 x g (16,000 rpm) for 1 minute. Dis-card the Receiver Tube with the filtrate.

8. Place the Spin Filter into an Elution Tube.

NOTE Avoid the carryover of Washing Solution residuals to the Elution Tubes.

Carefully open the cap of the Spin Filter and dry the Spin Filter by in-cubation at 60 °C for 10 minutes in a thermal mixer.

9. Add 50 μl Elution Buffer to the Spin Filter. Incubate at room temper-ature for 1 minute. Centrifuge at 8,000 x g (10,000 rpm) for 1 minute. Two elution steps with equal volumes of Elution Buffer (e.g. 25 μl + 25 μl) might increase the yield of converted DNA.

NOTE The converted DNA can be eluted with a lower or a higher volume of Elu-tion Buffer (depends on the expected yield of converted DNA). Elution with lower volumes of Elution Buffer increases the final concentration of converted DNA. Store the converted DNA at 4 °C to 8 °C. For long time storage placing at -22 °C to -18 °C is recommended.

Troubleshooting


15 Troubleshooting

Problem / probable cause Comments and suggestions

Clogged Spin Filter

Insufficient lysis and/or too much starting material

Increase lysis time. Increase centrifugation speed. After lysis centrifuge the lysate to pellet unlysed mate-rial. Reduce amount of starting material. Increase volume of Lysis Solution BC and Proteinase K.

Low amount of extracted/converted DNA

Insufficient lysis Increase lysis time. Reduce amount of starting material. Overloading of Spin Filter reduces yield!

Incomplete elution Prolong the incubation time with Elution Buffer to 5 minutes or repeat elution step once again. Take a higher volume of Elution Buffer.

Insufficient mixing with Binding Solution GS

Mix sample with Binding Solution GS by pipetting or by vortexing prior to transfer of the sample onto the Spin Filter.

Decomposition of Conver-sion Reagent

Use new aliquot of Conversion Reagent.

High fragmentation of extracted/converted DNA

Decomposition of Desulfo-nation Buffer

Do not use once opened vials of Desulfonation Buffer (concentrate). Do not use ready-to-use Desulfonation Buffer after dedicated reaction is completed.

Desulfonation Buffer is becoming hazy after addition of ethanol

Desulfonation Buffer was not stored under appropri-ate conditions.

Store Desulfonation Buffer as described in the manual. Discard this tube and prepare new ready-to-use Desul-fonation Buffer from unopened Desulfonation Buffer (concentrate) vial.

Low concentration of extracted/converted DNA

Too much Elution Buffer Elute the converted DNA with lower volume of Elution Buffer (min. 20 μl).

Failed PCR amplification of template DNA from FFPE tissue

Insufficient lysis Increase lysis time. Increase Proteinase K concentration.

Related Products


16 Related Products

Name Amount Order No.

innuCONVERT Bisulfite Basic Kit 8 rxn 845-IC-10000008

40 rxn 845-IC-10000040

80 rxn 845-IC-10000080

innuCONVERT Bisulfite Body Fluids Kit 8 rxn 845-IC-30000008

40 rxn 845-IC-30000040

80 rxn 845-IC-30000080

PME free-circulating DNA Extraction Kit 10 rxn 845-IR-0003010

50 rxn 845-IR-0003050

blackPREP FFPE DNA Kit 10 rxn 845-BP-0020010

50 rxn 845-BP-0020050

250 rxn 845-BP-0020250

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