Methylamp™ Whole Cell Bisulfite Modification Kit
Contents
1. Methylamp Whole Cell Bisulfite Modification Kit Base Catalog P 1016 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE The Methylamp Whole Cell Bisulfite Modification Kit is specifically designed for DNA methylation research using minute amounts of starting materials including cells cultured in 96 well 384 well plates tissue section samples microdissection samples tissue biopsy and early embryonic cells oocytes These materials may not be suitable for DNA isolation prior to bisulfite modification The Methylamp Whole Cell Bisulfite Modification Kit can also be suitable for DNA modification directly using cells or tissues for increasing modified DNA yield saving time and reducing labor Eluted modified DNA by using the Methylamp Whole Cell Bisulfite Modification Kit is suited for real time MS PCR It is also suitable for all techniques currently used for the analysis of DNA methylation including conventional MS PCR bisulfite sequencing pyrosequencing and methylation microarray If you use the Methylamp Whole Cell Bisulfite Modification Kit for MSP the numbers of PCR cycles should be greater than 45 The amount of starting materials for each modification can be 100 20000 cells or 1 ug 100 ug tissues or 0 2 2 mm tissue section samples For optimal modification the amount should be 500 5000 cells or 5 20 ug tissues or 0 5 1 mm tissue section samples respectively Suitable lab coat disposable gloves and eye protect2. ul of W6 to create the W4 W4 W6 solution Vortex until solution is clear or saturated about 2 minutes Add 110 ul of the mixed W4 W5 W6 solution to each PCR tube containing the sample Place the tube in a thermal cycler with heated lid and program the thermal cycler as followed 99 C for 20 minutes 65 C for 90 minutes 99 C for 10 minutes Modified sample can then be held at 25 C in the thermal cycler up to 4 hours without loss of performance Place a spin column into a 2 ml collection tube Add 200 ul of W7 to the column Transfer the sample from step 4 to the column containing W7 followed by adding 100 ul of 100 isopropanol to the column Sit for 2 minutes at room temperature and centrifuge at 12 000 rpm for 20 seconds Remove the column from the collection tube and discard the flowthrough Replace column to the collection tube Add 200 ul of 70 ethanol to the column and centrifuge at 12 000 rpm for 25 seconds Add 10 ul of W6 to 1 ml of 90 ethanol and mix to create the W6 ethanol solution Add 50 ul of the mixed W6 ethanol solution DNA cleaning solution to the column Sit for 10 minutes at room temperature then centrifuge at 12 000 rpm for 20 seconds Add 200 pl of 90 ethanol to the column centrifuge at 12 000 rpm for 20 seconds Remove the column from the collection tube and discard the flowthrough Replace column to the collection 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3
3. 956 m E mail info epigentek com m Web www epigentek com Printed 2015 09 01 Epigentek Group Inc All rights reserved Products are for research use only P 1016 tube Add 200 ul of 90 ethanol to the column again and centrifuge at 12 000 rom for 40 seconds Place the column in a new 1 5 ml vial Add 8 18 ul of W8 depending on the amount of starting materials directly to the column filter Centrifuge at 12 000 rpm for 20 seconds to elute modified DNA Modified DNA is now ready for methylation amplification or storage at 20 C for up to 2 months TROUBLESHOOTING DNA is Poorly Modified 1 Insufficient cell tissue lysis 2 Template contains high GC region or secondary structure 3 Thermal cycling condition is incorrect 4 Bisulfite reaction components are not mixed correctly 5 Insufficient DNA cleaning 6 Incorrect storage of W4 W5 W6 solution Elution Contains No or Little DNA 1 Poor starting material quality Ex FFPE sample contains fragmented DNA 2 Too little starting material ex lt 50 cells 3 W7 DNA Binding Buffer is not added into the sample 4 DNA cleaning solution is prepared incorrectly at step 7 of the protocol 5 The column is not washed with 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only
4. Increase incubation time to 60 90 minutes at 65 C at step 3 Increase bisulfite reaction time 65 C to 120 minutes at step 4 Check if the thermal cycling condition is set according to the protocol Ensure that each component is added correctly Ensure that a sufficient amount of W6 is added into 90 ethanol Ensure that W4 W5 W6 solution is stored at 20 C and for no more than 2 weeks Check if starting material is good in quality Increase starting material Ensure that W7 is added as described in step 5 Ensure that W6 is added into 90 ethanol Ensure that wash solution is 90 ethanol Page 6 Printed 2015 09 01 P 1016 90 ethanol 6 Sample is not completely passed through the filter Elution Contains Both Unmodified and Modified DNA 1 Amount of cells tissues used is out of recommended range to recommended range 2 Template with high G C content minutes in step 4 Poor Methylation Specific PCR Products 1 PCR components are not sufficiently added RELATED PRODUCTS P 1001 P 1002 P 1008 P 1010 P 1014 Methylamp DNA Modification Kit Methylamp Coupled DNA Isolation amp Modification Kit Methylamp 96 DNA Modification Kit Methylamp One Step DNA Modification Kit Methylamp Global DNA Methylation Quantification Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek
5. com Epigentek Group Inc All rights reserved Products are for research use only Increase centrifuge time to 1 minute at steps 5 9 Adjust the amount of starting cells tissues Increase bisulfite reaction time 65 C to 120 Check if all PCR components were added Page 7 Printed 2015 09 01 P 1016
6. he column to effectively remove residual sodium bisulfite and salts Modified DNA can then be eluted and stably stored at 20 C for up to 2 months 46 a 44 a Whole cell DNA modification amp a E 50 cells gt Capture and cleaning E 5000 cells of modified DNA 0 5 ng DNA 50 ng DNA 6 Elution of modified DNA 6 PCR sequencing hi Cycle Threshold CT P 1016 P 1010 Schematic Procedure for Using the Methylamp The different amounts of MCF 7 cells or DNA isolated Whole Cell Bisulfite Kit from MCF 7 cells were modified using the MethylampT Whole Cell Bisulfite Modification Kit or MethylampT One Step DNA modification kit respectively 10 ul of modified DNA were eluted and 2 ul of elution were used in real time PCR A pair of primers and a probe designed to amplify both methylated and unmethylated alleles of B actin PROTOCOL Note Always cap spin columns before placing them in the microcentrifuge Before starting prepare the following required solution not included 70 ethanol 90 ethanol and 100 isoprapanol Add 50 ul of W2 to W1 for P 1016 40 or add 100 ul of W2 to W1 for P 1016 80 to create the W1 W2 solution Vortex until solution is clear Collect the samples For adhesive cultures cells are detached by trypsinization and collected by centrifugation Add 10 ul of W3 to re suspend the cells and transfer into a 0 2 ml PCR tube For body fluids such as cerebro spinal fluid ascite sal
7. ion are required when working with the kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2015 09 01 P 1016 KIT CONTENTS Components W1 Digestion Powder W2 Digestion Solution W3 Cell Collection Buffer W4 DNA Modification Powder W5 DNA Modification Buffer Ws Balance Buffer W7 DNA Binding Buffer W8 Modified DNA Elution F Spin Column F Collection Tube User Guide SHIPPING amp STORAGE 40 samples P 1016 40 1 vial 0 1 ml 1 ml 4 vials 5 ml 0 4 ml 14 ml 1 ml 40 40 80 samples P 1016 80 1 vial 0 2 ml 2 mi 8 vials 10 ml 0 8 ml 28 ml 2 mi 80 80 The kit can be stored at room temperature 20 22 C away from light with the exception of component W1 Digestion Powder for 6 months from the shipping date Upon receipt W1 should be stored at 20 C or stored at 4 C as soon as it is dissolved in W2 up to 6 months Each vial of W4 can be used for 10 sample treatments The prepared W4 W5 W6 solution should be used immediately unless it is stored at 20 C away from light up to one week Frozen W4 W5 W6 solution must be thawed at room temperature and vortexed for 2 minutes prior to use MATERIALS REQUIRED BUT NOT SUPPLIED O Thermal cycler with heated lid Pipettes and pipette ti
8. ion of gene expression tumorigenesis and other genetic and epigenetic diseases such as cancer There have been many methods for the detection of DNA methylation Most of them require a bisulfite based DNA modification before starting methylation assays such as MSP sequencing restriction analysis and others The bisulfite based DNA modification is used to discriminate between cytosine and methylated cytosine in which bisulfite salt converts cytosine residues to uracil in single stranded DNA while methylated cytosine remains the same All current methods for DNA modification need to use isolated DNA as starting material which leads to the inability to achieve enough moditied DNA in tiny amounts of tissue or cell samples It is common that only minute amounts of tissue or cell samples can be available in biomedical research high throughput biomarker drug screening and pathological diagnosis These kinds of samples may include tissue biopsy microdissection samples cells contained in body fluids cells cultured in 96 and 384 well plates and early embryonic cells oocytes Thus direct DNA modification from whole cells or tissues would give an advantage to efficiently utilize these kinds of samples It could also generate a greater yield of moditied DNA because of avoiding DNA loss caused by DNA isolation purification prior to bisulfite modification To address this problem the Methylamp Whole Cell Bisulfite Modification Kit provides a useful tool
9. iva and urine cells are simply collected by centrifugation Add 10 ul of W3 to re suspend the cells and transfer into a 0 2 ml PCR tube For tissue biopsy add 10 ul of W3 into a 0 2 ml PCR tube and then add the sample to the PCR tube containing W3 For early embryonic cells or oocytes add 10 ul of W3 into a 0 2 ml PCR tube and then directly collect the cells into the PCR tube containing W3 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2015 09 01 Epigentek Group Inc All rights reserved Products are for research use only P 1016 NE For tissues from fresh sections add 10 ul of W3 into a 0 2 ml PCR tube Remove the tissue area you need from slide 0 2 2 mm which represent about 200 2000 cells assuming the section is 5 um thick and add it into the PCR tube containing W3 For tissues from formalin fixed paraffin embedded tissue sections remove the paraffin first with deparaftin reagents according to your own successful protocols or according to the following procedures 1 Drop the slide into 100 xylene at room temperature for 5 minutes Repeat once with new xylene 2 Drop the slide into 100 ethanol 95 ethanol and 70 ethanol for 5 minutes each Air dry the slide Cut the tissue area you need from the slide 0 2 2 mm and add it into the PCR tube containing 10 ul of W3 For microdissection samples from fresh o
10. ps 1 5 ml microcentrifuge tubes 0 2 ml PCR tube 15 ml conical tube Ethanol 96 100 OHoadaada GENERAL PRODUCT INFORMATION Desktop centrifuge up to 14 000 rpm Usage Limitation The Methylamp kit is for research use only and is not intended for diagnostic or therapeutic application 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2015 09 01 P 1016 Product Warranty Epigentek guarantees the performance of all products in the manner described in our product instructions Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design Intellectual Property The Methylamp kit and method of use contain proprietary technologies by Epigentek Methylamp is a trademark of Epigentek Group Inc A BRIEF OVERVIEW DNA methylation involves the course in which DNA methyltransferases DNMTs transfer a methyl group from S adenosyl L methionine to the fifth carbon position of the cytosines Aberrant DNA methylation is mainly found in 5 CpG 3 dinucleotides within promoters or in the first exon of genes which is an important pathway for the repression of gene transcription in diseased cells It is well demonstrated that DNA methylation plays an important role in the regulat
11. r frozen tissue sections add 3 5 ul of W3 into the center of the cap of a 0 5 ml PCR tube Place the cap on the tissue to be microdissected and capture cells 100 1000 cells into the cap Remove the cap and place it back to the PCR tube containing 10 ul of W3 Note if cap is dried add an additional 3 5 ul of W3 into the cap before placing it back to the PCR tube Centrifuge at 12 000 rpm for 30 seconds to move the cells down to the solution in the tube Transter the solution containing cells into a new 0 2 ml PCR tube For microdissection samples from formalin fixed paraffin embedded tissue sections remove the paraffin first with deparatfin reagents according to your own successful protocols or according to the procedures described above Add 3 5 ul of W3 into the center of the cap of a 0 5 ml PCR tube Place the cap on the tissue to be microdissected and capture cells 400 1000 cells into the cap Remove the cap and place it back to the PCR tube containing 10 ul of W3 Note if cap is dried add additional 3 5 ul of W3 into the cap before placing it back to the PCR tube Centrifuge at 12 000 rpm for 30 seconds to move the cells down to the solution in the tube Transfer the solution containing cells into a new 0 2 ml PCR tube Add 1 ul of the mixed W1 W2 solution to the PCR tubes containing the samples and place the tubes in a thermal cycler with the program of 65 C for 45 minutes Meanwhile add 1 ml of W5 to 1 vial of W4 followed by adding 60
12. to modify DNA directly from the cells or tissues The kit has the following features e Fast results streamlined 3 hour procedure from cells tissues to moditied DNA e Completely converts unmethylated cytosine into uracil modified DNA gt 99 5 e The lowest degradation of DNA in the modification process more than 90 of DNA loss can be prevented with unique DNA protecting buffer PRINCIPLE amp PROCEDURE The Methylamp Whole Cell Bisulfite Modification Kit contains all reagents required for bisulfite conversion directly on a cell or tissue sample The kit allows DNA to be isolated from cells or tissues denatured and bisulfite modified simultaneously in the same tube with the specific reaction buffer under the thermodynamic condition In the modification process bisulfite reagent reacts specifically with single stranded DNA thereby deaminating cytosine and creating a uracil residue 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2015 09 01 P 1016 The unique DNA protection reagents contained in the modification buffer can prevent the chemical and thermophilic degradation of DNA in the bisulfite treatment The non toxic modified DNA capture buffer enables DNA to tightly bind to the column filter thus DNA cleaning can be carried out on t
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