The instructions for use must be r and followed
Contents
1. 10 OT EEE EEE 10 194 3 OAS BU CG reset 10 toii DNAN A ia ING ee AAEE ee er ee ee 10 DO ENUM sacs ceitnc voce erect E E 11 10 1 6 Storage or extracted DNArersusen ee 11 10 2 Bisan E CONV CISION ee 11 10 2 1 Bisu ulite DNA Purnlcationsnsen ee 12 10 22 BAIDD a or E E N 12 105 ENa a E EE 13 1024 NaN Sierra 13 102 3 Br NN IS ee ET Teer 13 1026 Dogs ee E 13 10 27 ERO ee een ee rue 13 10 2 8 Storage of bisulfite treated DNA 02220002ss0nnssnnnssnnnnnnnnnnnnnnnnnnnnsnnnsnnnnssnnnnnnnnn 13 Ei MOMMIES OTIC Oleg ogre vag ee a ee 14 12 Limitations OF the Procedure susanne 14 Is PEROrmanee Cal ACIS IGS nenne 14 14 ROTO CS acca crc cece ace eaves ee ease raeee EEE A E 14 152 COMICS UDDOEL ernennen rere rrr er re rns 15 pro colon lasma DNA Preparation Kit Page 2 of 15 pro colon Ines for lice Instructions tor Use Plasma DNA Preparation Kit 1 Important Note Important Users should familiarize themselves with the instructions contained in this booklet and the components of the Epi proColon Plasma DNA Preparation Kit prior to use Meaning of Symbols Consult Instructions for Use Order Number HERS In Vitro Diagnostic Medical Device g DO Lot Number g Expiration Date Manufacturer Store at indicated Temperature Number of Tests El lt lt E RK a N ys Keep away from Sunlight Page 3 of 15 epi lasma DNA Preparation Kit 2 Intended Use The Epi proCol2. pro epl Plasma DNA Preparation Kit The instructions for use must be read carefully prior to use and followed precisely to achieve reliable results li IFU 0002GB rev 6 copyright March 2011 Epigenomics AG M5 01 001 v 24 Epigenomics AG e D g e N O m C S 10178 Berlin Germany s Instructions for Use SP Content le IOP ANEO E E E ER E E EA E 3 2 11 e E ee A E eee eee ee ee ere 4 De PROCUCE DESCHP ION De ee 4 4 Technological Principles urn 4 9 OT Se spn A AE ET P OEE tains O E 5 6 Instruments Supplies and Reagents Required but not Provided essessessenssessseessse 5 7 SAO e EEEO ee 7 Oe OEA Sl a 8 rrer AA EEEO 8 9 Test Materials and Preparation for AnallySis cccsssecceeecceescceeecceeeecceeeceeeecceeeeenseeeeees 8 9 1 Preparation of Working SolutionsS esssssssssesssesssessssesssessersserssessseresersseessersserssessseress 8 9 1 1 Preparation of Epi proColon Proteinase Solution u 0ssssennnsennnsennnnnenennn 8 9 1 2 Preparation of Epi proColon Carrier Solution 0 ss40ssssonnsennnnennnennnnnen nen 8 91 3 Preparation of Epi proColon Bis Reagent A Solution us0sssonssnnnnennnnnnn 9 JA SAMPE PrE Para ION nET TOA E A OR 9 ae PAPE a HOM nee een J 10 Experimental Dion E EE N 10 10 1 PY SUS and EU A O en A EEA EN E 10 10 1 1 Thawing of plasma only required if frozen material is used
3. 254 or equivalent Safe Lock reaction tubes 0 5 ml PCR tubes e g Eppendorf AG catalog no 0030 121 023 or equivalent Safe Lock reaction tubes 1 5 ml PCR tubes e g Eppendorf AG catalog no 0030 120 086 or equivalent Safe Lock reaction tubes 2 ml PCR tubes e g Eppendorf AG catalog no 0030 120 094 or equivalent Pipette tips with aerosol barrier e g VWR International GmbH 612 1430 KOEHK 0518 613 2658 or equivalent Tube rack for 50 ml tubes and 2 ml tubes VWR International GmbH 50 ml catalog no 211 0200 2 ml catalog no 211 0213 or equivalent Page 5 of 15 SP pro colon lasma DNA Preparation Kit Disposable gloves Magnetic Separator chemagic Stand 50 k Typ B Chemagen catalog no 303 Magnetic Separator chemagic Stand 2 x 12 Chemagen catalog no 300 Rotating shaker SB 2 Carl Roth GmbH Y549 1 with rotating plate Carl Roth GmbH Y553 1 or equivalent Waterbath Gesellschaft f r Labortechnik GmbH GFL Type 1002 catalog no 163 9905902 or equivalent Vortex VWR International GmbH catalog no 444 1372 or equivalent Thermomixer comfort catalog no 5355 000 011 with dry heating block 2 ml Eppendorf AG catalog no 5362 000 019 or equivalent Rotilabo Floating rack for 50 ml centrifuge tubes Carl Roth GmbH catalog no P201 1 or equivalent Reference pipette sets 0 1 2 5 ul 0 5 10 ul 10 100 ul 100 1000 ul Eppendorf AG or equivalent 2 3 ml Pasteur single u
4. Solution Add 1 5 ml Epi proColon Purification 1 Note Epi proColon Carrier Solution can be mixed with Epi proColon Purification 1 before use to enable the handling of larger volumes This mixture can be stored at room temperature for up to 12 h Add 10 ul of thoroughly resuspended Epi proColon Magnetic Beads Note A homogeneous suspension of beads in Epi proColon Magnetic Bead Suspension is essential for proper performance Deviations from the specified amount of beads may lead to false results Close lid and vortex intensively for 10 sec Place tubes in a thermomixer and incubate at room temperature for 60 min while shaking at 1 400 rpm Remove tubes from thermomixer and set the thermomixer temperature to 55 C 10 Pulse spin to remove drops from lid Place tubes into a magnetic separator and allow the beads to accumulate for 2 min 2 Using a fresh pipette remove as much liquid as possible Page 12 of 15 pro colon Ines for lice Instructions tor Use Plasma DNA Preparation Kit 10 2 3 1st Wash Remove 2 ml tubes from the magnetic rack and transfer to a non magnetic rack 2 Add 300 ul Epi proColon Purification 2 3 Close lid and vortex briefly to resuspend the beads Pulse spin to remove drops from lid Place tubes into a magnetic separator and allow the beads to accumulate for 2 min Completely remove supernatant with a pipette 10 2 4 2nd Wash Repeat steps 1 to 6 from section 10 3 2 10 2 5 3rd Wash Re
5. The Epi proColon Plasma DNA Preparation Kit is manufactured by Epigenomics AG Kleine Pr sidentenstra e 1 10178 Berlin Germany using components from Chemagen Biopolymer Technologie AG Arnold Sommerfeld Ring 52499 Baesweiler Germany For further information and support send an e mail to support products epigenomics com or call 49 30 24345 222 Page 15 of 15
6. ains Sodium sulfite and Sodium metabisulfite Sodium disulfite R22 31 41 S26 39 Epi proColon Bis Reagent B contains Trolox Tetrahydrofurfurylalcohol R36 39 Epi proColon Extraction 4 Epi proColon Purification 4 Epi proColon Magnetic Beads and Epi proColon Carrier do not contain harmful components R phrases R 9 Explosive when mixed with combustible material R 11 Highly flammable R 20 21 22 Harmful by inhalation in contact with skin and if swallowed R 22 Harmful if swallowed R 31 Contact with acids liberates toxic gas R 32 Contact with acids liberates very toxic gas R 36 Irritating to eyes R 36 37 38 Irritating to eyes respiratory system and skin R 41 Risk of serious damage to eyes R 42 43 May cause sensitisation by inhalation and skin contact R 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Page 7 of 15 epi P lasma DNA Preparation Kit S phrases S 7 Keep container tightly closed S 13 Keep away from food drink and animal feedingstuffs S 16 Keep away from sources of ignition No smoking S 22 Do not breathe dust S 24 25 Avoid contact with skin and eyes 5 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S 36 37 Wear suitable protective clothing and gloves S 37 Wear suitable gloves S 39 Wear eye face protection S 45 In case of accident or if you feel unwell seek medical advice imm
7. ated DNA is not used immediately store material at 2 to 8 C for up to 24 hours or at 15 to 25 C for up to 3 days Page 13 of 15 epi lasma DNA Preparation Kit 11 Quality Control The Epi proColon Work Flow Control Kit M5 01 003 contains Epi proColon Positive and Negative Controls These controls must be included with each assay run to monitor the successful execution of the workflow to ensure validity of test results The Epi proColon Positive Negative Control values must be within the validity limits specified in the Instruction For Use of the Epi proColon real time PCR Kit M5 01 002 If a control is out of its specified range the associated test results are invalid must not be reported and the patients must be retested If laboratory quality control procedures require more frequent use of controls to verify test results follow those procedures 12 Limitations of the Procedure This product has been validated for use with human EDTA plasma Using the product on other materials has not been validated and is not recommended This product has been validated for use with the Epi proColon real time PCR Kit M5 01 002 Performance with other downstream applications has not been established We recommend using only single use pipettes and pipette tips to prevent cross contamination of patient samples Haemolytic samples should not be used as the impact of hemolysis on test results is not known 13 Performance Chara
8. cteristics The Epi proColon Plasma DNA Preparation Kit has been tested in combination with the Epi proColon real time PCR kit M5 01 002 on control materials provided by the Epi proColon Work Flow Control Kit M5 01 003 on technical materials containing buffered concentrations of DNA on plasma pools prepared from human EDTA plasma samples and on individual plasma samples The Epi proColon Plasma DNA Preparation Kit provided bisulfite converted DNA suitable for real time PCR in more than 99 of the cases Performance of the Epi proColon Plasma DNA Preparation Kit has been established with the Epi proColon real time PCR Kit For details the user is referred to the Instructions for Use of the Epi proColon real time PCR Kit 14 References De Vos T et al Circulating methylated SEPT9 DNA in Plasma is a Biomarker for Colorectal Cancer Clinical Chemistry 55 7 1337 46 2009 2 Lofton Day C et al DNA methylation biomarkers for blood based colorectal cancer screening Clinical Chemistry 54 2 414 423 2008 Gruetzmann R et al Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay PLoS ONE Volume 3 Issue 11 e3759 2008 Model F et al Identification and validation of colorectal neoplasia specific methylation markers for accurate classification of disease Mol Cancer Res 5 153 163 2007 Page 14 of 15 epi colon Instructions for Use Plasma DNA Preparation Kit 15 Contact Support
9. ediately show this label S 61 Avoid release to the environment Refer to special instructions and safety data sheet 8 Storage and Stability 25 C Deo Store all reagents of the Epi proColon Plasma DNA AY Preparation Kit at 18 to 25 C in the dark 18 C j 15 C For long term storage up to 3 months dissolved Carrier Proteinase and Bis Reagent A should be aliquoted and stored at 15 to 25 C Avoid 25 C freeze thaw cycles Reagents provided with the Epi proColon Plasma DNA Preparation Kit are stable until the expiration date when stored and handled as directed Do not use material past expiration date Do not mix components from different kit lots Note If Epi proColon Extraction 1 contains precipitates proceed as follows Heat Epi proColon Extraction 1 in a waterbath at 50 5 C for 10 min and shake gently Repeat heating and shaking until the precipitate is completely dissolved Bring Epi proColon Extraction 1 to room temperature 18 25 C before use The brown color of the Epi proColon Bis Reagents is normal and has no impact on their performance 9 Test Materials and Preparation for Analysis 9 1 Preparation of Working Solutions 9 1 1 Preparation of Epi proColon Proteinase Solution Add 860 ul water molecular biology grade to a tube containing Epi proColon Proteinase Vortex intensively for 1 min Repeat vortexing procedure until all material is dissolved 9 1 2 Preparation of Epi proCo
10. ers and probes used in a subsequent PCR reaction are sensitive to these base pair changes and will discriminate between methylated and unmethylated sequences 4 Technological Principles The extraction of genomic DNA contained in patient plasma is based on the binding of free floating genomic DNA to magnetic particles which are then magnetically separated from the plasma Remaining impurities are removed from the magnetic particles during the washing steps In the elution step purified DNA is removed from the magnetic particles by dissolving in elution buffer The eluate containing genomic DNA is then subjected to a chemical reaction that specifically alters unmethylated cytosine residues within the DNA Bisulfite treatment is utilized as the method of choice for analyzing DNA methylation This method allows various downstream applications as the sequence context remains intact The conversion is based on the nucleophilic addition of a bisulfite ion to a cytosine target and a subsequent deamination reaction to yield uracil sulfonate while 5 methylcytosine methylated cytosine does not undergo the deamination reaction and remains unchanged Complete conversion of unmethylated cytosines and high DNA integrity after treatment are the critical parameters for sensitive measurement of DNA methylation Incomplete denaturation prior to treatment and harsh chemical conditions during the reaction have been identified as the most common pitfalls of the proced
11. lon Carrier Solution 1 Add 205 ul Epi proColon Carrier Diluent to a tube containing Epi proColon Carrier Vortex intensively for at least 2 min Page 8 of 15 eDIE olon Instructions for Use Plasma DNA Preparation Kit 9 1 3 Preparation of Epi proColon Bis Reagent A Solution I Add 800 ul of water to a tube containing Epi proColon Bis Reagent A 2 Vortex intensively until residuals are dissolved partially Add additional 800 ul water 4 Vortex intensively for 3 min Repeat vortexing procedure until all material is dissolved Note The Epi proColon Bis Reagent A is hardly soluble therefore it is possible to gently heat the solution 45 to 55 C 9 2 Sample Preparation Use a Vacutainer K EDTA 10 ml tube S Monovette Haematology K EDTA 9 0 ml tube or S Monovette CPDA 8 5 ml tube for drawing blood Do not freeze blood samples Preparation of plasma may be performed up to 24 hours after collection if blood samples were stored continuously at 2 to 8 C If S Monovette CPDA 8 5 ml tubes are used blood samples may be stored for up to 48 h at 2 to 25 C 9 3 Plasma Preparation Disable the brake function in the centrifuge to prevent disruption of the cell layer 2 Centrifuge the blood in Vacutainer or S Monovette tubes for 12 min at 1350 150 g If centrifuge uses RPM revolutions per minute refer to the centrifuge user manual for a conversion table Remove tube from centrifuge Discard the sample if hemolysi
12. move 2 ml tubes from the magnetic rack and transfer to a non magnetic rack 2 Add 300 ul Epi proColon Purification 3 3 Close lid and vortex briefly to resuspend the beads Pulse spin to remove drops from lid Place tubes into a magnetic separator and allow the beads to accumulate for 2 min Completely remove supernatant with a pipette Spin for 30 sec at 1 000 150 g to remove drops from lid Place tubes into a magnetic separator and allow the beads to accumulate for 2 min Remove residual liquid with a pipette 10 2 6 Drying Place tubes with lids open into the pre heated thermomixer and incubate for 5 min at 552 C Note Do NOT over dry Longer incubation times may cause lower DNA yields 2 Remove 2 ml tubes from the thermomixer and transfer to a non magnetic rack 10 2 7 Elution Add 55 ul Epi proColon Purification 4 and vortex briefly to resuspend the beads Place tubes into a thermomixer and incubate at 55 2 C for 15 min while shaking at 1 400 rpm Pulse spin to remove liquid from lid Place tubes into a magnetic separator Allow the beads to accumulate for 2 min Extend separation time if any beads are present in the elution buffer Transfer the complete eluate into a multiwell plate Alternatively 8 stripe tubes or PCR reaction tubes or 1 5 ml Safe Lock tubes can be used Following elution the bisulfite treated DNA can be transferred into 10 2 8 Storage of bisulfite treated DNA If extracted bisulfite tre
13. on Plasma DNA Preparation Kit contains reagents for the preparation of bisulfite converted DNA from human EDTA plasma suitable for real time PCR for in vitro diagnostic purposes The Epi proColon Plasma DNA Preparation Kit includes reagents for DNA extraction DNA bisulfite conversion and post bisulfite DNA purification Performance of the Epi proColon DNA Plasma Preparation Kit has been validated for use with the Epi proColon real time PCR Kit M5 01 002 3 Product Description The Epi proColon Plasma DNA Preparation Kit is used for the extraction of DNA from 3 5 ml plasma followed by bisulfite conversion of the purified DNA After a final purification step the DNA is suited for the analysis of methylation patterns by real time PCR amplification The principle of detection of methylated DNA is comprised of three steps starting with a plasma sample that is prepared from whole blood using standard techniques DNA methylation occurs predominantly on a CpG dinucleotide a cytosine nucleotide followed by a guanine nucleotide DNA contained within the plasma sample is extracted using magnetic bead based nucleic acid extraction The extracted DNA is then chemically converted to reveal the methylation status using sodium bisulfite This chemical treatment converts unmethylated cytosines in CpG dinucleotides to uracils while the methylated cytosines remain unchanged resulting in a single base pair change at each unmethylated cytosine The primers block
14. owing order 70 ul Epi proColon Magnetic Beads freshly suspended by vortexing for 30 sec 10 ml Epi proColon Extraction 2 Vortex intensively for 10 sec Place 50 ml tubes into a rotating shaker and incubate at room temperature for 60 5 min Note A homogeneous suspension of beads in Epi proColon Magnetic Bead Suspension is essential for proper performance Deviations from the specified amount of beads may lead to false results 10 1 4 DNA Washing Place 50 ml tubes into the magnetic rack and allow beads for 5 min to form a pellet Discard supernatant Ensure that the supernatant is completely removed Add 1 5 ml Epi proColon Extraction 3 and vortex intensively for 10 sec Ensure that the magnetic beads are completely suspended Transfer bead suspension into a 2 0 ml Safe Lock tube with a Pasteur single use pipette Pulse spin the 2 ml tube to remove drops from the lid Place tubes into a magnetic separator Allow the beads to accumulate for 2 min Discard supernatant with a pipette to the extent possible while taking care not to remove beads Place tubes into a centrifuge and spin for 10 sec at 1 000 150 g Place tubes into a magnetic separator Allow the beads to accumulate for 2 min and remove residual washing solution Page 10 of 15 e HA Instructions for Use Plasma DNA a Kit 10 1 5 Elution l Transfer tubes into a non magnetic rack 2 Add 100 ul Epi proColon Extraction 4 to each tube and vortex intensivel
15. s bright red plasma is observed In this case a new blood sample must be taken Use a Pasteur single use pipette to transfer plasma from the collection tube to a 15 ml centrifuge tube Centrifuge plasma in the 15 ml centrifuge tube for 12 min at 1350 150 g Using a new bulb pipette transfer 3 5 ml plasma from the 15 ml centrifuge tube to a 50 ml polypropylene centrifuge tube with conical bottom Start DNA extraction with Epi proColon Plasma DNA Preparation Kit Alternatively store plasma samples at 2 to 8 C for up to 18 hours or at 15 to 25 C for up to 7 days Page 9 of 15 SP lasma DNA Preparation Kit pro colon 10 Experimental Protocol 10 1 Lysis and Extraction 10 1 1 Thawing of plasma only required if frozen material is used Thaw frozen plasma for 30 min at room temperature 10 1 2 Lysis Add the following to a 50 ml tube 3 5 ml blood plasma sample or Epi proColon Positive Control or Epi proColon Negative Control 3 5 ml Epi proColon Extraction 1 5 ul Epi proColon Carrier Solution 20 ul Epi proColon Proteinase Solution The total volume will be 7 ml Note Prior to use briefly shake Epi proColon Extraction 1 tube to check for precipitates If precipitates are present follow instructions detailed in the Note in Section 8 Mix by vortexing for 10 sec and place tubes into a 56 2 C waterbath for 10 min 10 1 3 DNA Binding Remove 50 ml centrifuge tubes from waterbath and add in the foll
16. se pipette Carl Roth GmbH catalog no EA56 1 or equivalent 6 ml Pasteur single use pipette VWR International GmbH catalog no 612 4464 or equivalent Multipette Plus Eppendorf AG catalog no 4981 000 019 or equivalent Mastercycler Eppendorf AG catalog no 5331 000 018 or equivalent with heatblock suitable for 0 5 ml reaction tubes Centrifuge 5417 R Eppendorf AG catalog no 5407 000 317 or equivalent Epi proColon Work Flow Control Kit M5 01 003 Page 6 of 15 ne K COIOI Inn en tr En a a J22 NSTFUCTIONS TOF U sc Plasma DNA Preparation Kit 7 Safety Information Always wear a laboratory coat and disposable gloves Clean contaminated surfaces with water Epi proColon Extraction 2 contains Sorbitan Sesquioleate Sodium x perchlorate and Ethanol R9 11 22 13 S16 Epi proColon Extraction 3 contains Sorbitan Sesquioleate Sodium x perchlorate and Ethanol R9 11 22 S13 16 Epi proColon Extraction 1 contains Guanidine Isothiocyanate R20 21 22 32 52 53 S13 36 37 S61 Epi proColon Purification 1 contains Sorbitan Sesquioleate Sodium e x perchlorate and Ethanol R9 11 22 S7 16 Epi proColon Purification 2 contains Ethanol R11 S7 16 Epi proColon Purification 3 contains Ethanol R11 S7 16 Epi proColon Proteinase R36 37 38 42 43 S22 24 25 37 45 Epi proColon Carrier Diluent contains Guanidine isothiocyanate R20 21 22 32 52 53 S13 36 37 61 Epi proColon Bis Reagent A cont
17. t to heat the lid of the Mastercycler to 105 C during the entire incubation procedure to avoid evaporation of reaction components Converted DNA can be left in the Mastercycler overnight without any loss of performance Do not freeze the reaction mixture Page 11 of 15 epi lasma DNA Preparation Kit Table 1 Bisulfite Conversion Thermal Cycler Program Step No Step Time Temperature 1 Initial Denaturation 5 min 96 C 2 Incubation 25 min 50 C 3 Denaturation 5 min 96 C 4 Incubation 85 min 1h 25 min 50 C 5 Denaturation 5 min 96 C 6 Incubation 295 min 4h 55 min 50 C 7 Hold lt 14 h 20 C 10 2 1 Bisulfite DNA Purification Prepare the following tubes for each sample One 2 ml Safe Lock reaction tube per sample for the washing steps One 1 5 ml Safe Lock reaction tube per sample for the elution step 10 2 2 Binding Step Note The DNA yield after purification strongly depends on the correct amount of magnetic beads being added to the reaction mixture Resuspend Epi proColon Magnetic Beads by vortexing intensively for 30 sec Repeat suspension procedure of Epi proColon Magnetic Beads after every 6 samples to ensure the same amount of magnetic particles in each DNA extraction solution Vortex 0 5 ml Safe Lock tubes containing the bisulfite reaction mixture intensively for 10 sec Pulse spin to remove drops from lid Transfer the complete reaction mixture into a 2 0 ml Safe Lock tube Add 1 ul Epi proColon Carrier
18. ure The work flow protocol maintains DNA in a single stranded form and uses gentle chemical conditions to ensure complete bisulfite conversion high DNA yields and low degradation of the converted DNA After purification of the DNA by binding to magnetic particles and washing to remove impurities the DNA is suited for use in downstream applications Page 4 of 15 epi lasma DNA Preparation Kit 5 Contents The Kit contains reagents for 24 preparations of DNA from plasma Reagent Containers Epi proColon Extraction 1 1 bottle Epi proColon Extraction 2 2 bottles Epi proColon Extraction 3 1 bottle Epi proColon Extraction 4 2 tubes Epi proColon Magnetic Beads 2 tubes Epi proColon Carrier 1 tube Epi proColon Carrier Diluent 1 tube Epi proColon Proteinase 1 tube Epi proColon Bis Reagent A 3 tubes Epi proColon Bis Reagent B 1 tube Epi proColon Purification 1 1 bottle Epi proColon Purification 2 1 bottle Epi proColon Purification 3 1 bottle Epi proColon Purification 4 1 tube Volume 90 ml 130 ml each 39 ml 1300 ul each 1100 ul 210 ul 6 Instruments Supplies and Reagents Required but not Provided Vacutainer EDTA Tube Becton Dickinson catalog no 367525 or S Monovette Haematology K EDTA 9 0 ml Sarstedt catalog no 02 1066 001 S Monovette CPDA 8 5 ml Sarstedt catalog no 01 1610 001 Water molecular biology grade 50 ml polypropylene centrifuge tubes with conical bottom e g Sarstedt catalog no 62 547
19. y until the magnetic beads are completely resuspended Place tubes into a thermomixer and incubate at 65 2 C for 15 min while shaking at 1 400 rpm Pulse spin for 10 sec to remove drops from lid Place tubes into a magnetic separator Allow the beads to accumulate for 2 min Extend separation time if any beads are present in the supernatant Transfer the complete supernatant into a 0 5 ml Safe Lock tube Elution volume will be 100 ul 10 1 6 Storage of extracted DNA If extracted DNA is not used immediately store material at 2 to 8 C for up to 24 hours or at 15 to 25 C for up to 3 days 10 2 Bisulfite Conversion Note Always add reagents in the order listed below Do not pre mix Epi proColon Bis Reagent A Solution and Epi proColon Bis Reagent B Solution before addition to the DNA Do not place dissolved Epi proColon Bis Reagent Solutions on ice Do not carry over beads into the bisulfite reaction Prepare bisulfite reactions in 0 5 ml PCR tubes according to and in the order listed below l Add 190 ul Epi proColon Bisulfite Reagent A Solution Add 30 ul Epi proColon Bisulfite Reagent B Solution Note The total volume is approximately 320 ul Close the PCR tubes and vortex the bisulfite reactions thoroughly for 20 sec Pulse spin the tubes to remove liquid from the lid Place the PCR tubes into the deep wells of the Mastercycler heating plate Use the Mastercycler program listed in Table 1 Note It is importan
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