Contents Introduction Storage and Stability Binding Capacity
Contents
1. Add 25 ul of OB Protease and vortex to mix well Incubate at 55 C in a shaking waterbath to effect complete lysis If no shaking waterbath is available vortex the sample every 20 30 minutes Lysis time depends on amount and type of tissue but is usually under 3 hours One can allow lysis to proceed overnight The volume of OB Protease or proteinase K used will need to be adjusted based on amount of starting material use 50 ul for a 60 mg tissue sample OPTIONAL Certain tissues such as liver have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point Add 5pl assuming a sample size of 30 mg RNase A 25 mg ml and incubate at room temperature for 2 5 minutes Proceed with the tissue protocol Centrifuge for 5 min at 10 000 x g to pellet insoluble tissue debris Carefully aspirate the supernatant and transfer to a sterile micro centrifuge tube leaving behind any insoluble pellet Add 220 ul Buffer BL and vortex to mix Incubate at 70 C for 10 min A wispy precipitate may form on addition of Buffer BL but does not interfere with DNA recovery Adjust the volume of Buffer BL required based on amount of starting material Add 220 ul absolute ethanol room temperature 96 100 and mix thoroughly by vortexing at maxi speed for 15 seconds Adjust the volume of ethanol if greater than 30 mg tissue is used If precipitation can be seen at this point2. gt 12 000 x g for 2 min to dry the column This step is crucial for ensuring optimal elution in the following step Place the column into a sterile 1 5 ml microfuge tube and add 50 200 ul of preheated 70 C Elution Buffer Allow tubes to sit for 3 min at room temperature To elute DNA from the column centrifuge at 10 000 x g for 1 min Repeat the elution with a second 100 200 ul of Elution Buffer Note Each 100 200 ul elution typically yields 60 70 of the DNA bound to the column Thus two elutions generally give 90 However increasing elution volume reduces the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 ul to 100 ul Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 ul greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon addition of Elution Buffer If necessary the DNA can be concentrated Add sodium chloride to a final concentration of 0 1 M followed by 2X volume of absolute 100 ethanol Mix well and incubate at 20 C for 10 min Centrifuge at 10 000 x g for 15 min and discard supernatant Add 700 pl of 80 ethanol and centrifuge at 10 000 x g for 2 min Discard supernatant air dry the pellet 2 min and resuspend DNA in 20 ul sterile deionized water or 10 mM Tris HCl pH 8 0 B E Z N A Protocol for Cultured Cells Have the following reagents an
3. than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digests LOW A 6o Aso ratio Poor cell lysis due to Repeat the procedure this time incomplete mixing making sure to vortex the sample with Buffer BL with Buffer BL immediately and completely May 25 2009 10 Problem Possible Cause Suggestions Ordering Information Incomplete cell lysis Increase incubation time with Buffer eee or protein TL and protease Ensure that no Product No Product Name Description degradation due to visible pieces of tissue remained i n Blood DNA Isolation Kits insufficient incubation D3392 01 02 Blood DNA Kit Isolation of total cellular DNA Samples are rich in After applying to column wash with from fresh and dry blood protein 300 pl of a 1 1 mixture of Buffer BL and ethanol and then with DNA Wash Buffer D3494 01 02 Blood DNA Midi Kit Isolation of total cellular DNA from up to 10ml blood No DNA eluted Poor cell lysis due to improper mixing with Buffer BL Mix thoroughly with Buffer BL prior to loading into HiBind column D2492 01 02 Blood DNA Maxi Kit Isolation of total cellular DNA from up to 20ml blood Poor cellcand or Tissue sample must be cut or minced D1192 01 02 E Z 96 Blood DNA Kit Isolation of total cellular DNA protein lysis in Buffer into small pieces Increase from fresh and dry blood
4. break the precipitation by pipetting up and down 10 times 10 11 12 13 14 Assemble a HiBind DNA column in a 2 ml collection tube provided Transfer the entire lysate from step 6 into the column including any precipitate that may have formed Centrifuge at 8 000 x g for 1 min to bind DNA Discard flow through liquid Optional If greater than 30 mg tissue is used repeat transfer the remaining lysate into the column and centrifuge as above Make sure that all of the lysate has pass through the column Place the column into a second 2 ml collection tube and wash by pipetting 500 ul of Buffer HB Centrifuge at 8 000 x g for 1 min Discard flow through liquid and 2ml collection tube Place the column into a second 2 ml collection tube and wash by pipetting 700 pl of DNA Wash Buffer diluted with ethanol Centrifuge at 8 000 x g for 1 min Discard flow through liquid and re use 2ml collection tube in next step Note that DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle or page 3 If refrigerated the diluted DNA wash buffer must be brought to room temperature before use Place the column back into the 2ml collection tube from step 10 wash the column with a second 700 ul of DNA Wash Buffer diluted with ethanol and centrifuge as above Discard flow through Place the column back into the same 2 ml collection tube centrifuge the empty column at maximum speed
5. with 96 Tk incubation time at 65 C with Buffer well plate TL to ensure that tissue is completely lysed Tissue DNA Kits Absolute ethanol not Before applying sample to column an added to Buffer BL aliquot of Buffer BL ethanol must be added See protocol above D3096 01 02 Micro Spin Genomic DNA Isolation of total cellular DNA Kit from micro samples No ethanol added to Wash Buffer Concentrate Dilute Wash Buffer with the indicated volume of absolute ethanol before use from tissue samples D1196 01 02 E Z 96 Tissue DNA Kit Isolation of total cellular DNA from tissue samples with 96 well plate Washing leaves colored residue in column Incomplete lysis due to improper mixing with Buffer BL Buffer BL is viscous and the sample must be vortexed thoroughly D5197 01 12 HP Tissue DNA Midi Kit Isolation of total cellular DNA from 500mg tissue samples No ethanol added to Dilute Wash Buffer with the indicated Wash Buffer volume of absolute ethanol before Concentrate use D5198 01 02 HP Tissue DNA Maxi Kit Isolation of total cellular DNA from 2 g tissue samples D3592 01 02 Forensic DNA Kit Isolation of genomic DNA from forensic samples OB collection paper included D3396 01 02 Tissue DNA Kit Isolation of total cellular DNA D3373 01 02 Mollusc DNA Kit Isolation of total cellular DNA If the above suggestions fail to resolve any problems you are having with th
6. Buffer BL and vortex to mix Incubate at 70 C for 10 min A wispy precipitate may form on addition of Buffer BL but does not interfere with DNA recovery Adjust the volume of Buffer BL required based on amount of starting material Add 220 ul absolute ethanol room temperature 96 100 and mix thoroughly by vortexing at maxi speed for 15 seconds If precipitation can be seen at this point break the precipitation by pipetting up and down 10 times Proceed Step 7 14 of E Z N A Protocol for tissue on Page 5 C E Z N A Protocol for Mouse Tails Snips Before Starting have the following ready Tabletop microcentrifuge and sterile 1 5 ml tubes Shaking waterbath set to 55 C Elution Buffer 0 5 ml per sample equilibrated to 70 C For each sample premix 200 pl Buffer BL with 200 pl absolute ethanol and vortex This can be prepared fresh or pre made and stored at room temperature Do not store this mixture for more than 1 month Have a shaking waterbath set to 55 C Absolute ethanol approximately 0 3 ml per sample RNase A Optional stock solution at 25 mg ml Bring frozen samples and OB Protease solution to room temperature and preheat an aliquot of Elution Buffer approximately 0 5 ml per sample at 70 C 1 Snip two pieces of mouse tail 0 2 0 5 cm in length place into a sterile 1 5 ml microcentrifuge tube and add 180 ul of Buffer TL If necessary cauterize the wound to stop bleeding Having appropriately ea
7. Contents Introduction Storage and Stability Binding Capacity Kit Contents Before Starting A E Z N A Protocol B E Z N A Protocol C E Z N A Protocol TOF TISSUS s sie 5 Seed gee eee seals PR oS See Oe for cultured cells a n ee et eee for Mouse Tails Snips 0 2 2 e eee ee eee D E Z N A Protocol for Paraffin Embedded Tissue 08 E E Z N A Tissue DNA Vacuum spin protocol 000 ee eee Determination of Yie Id and Quality es 62 sues Se ea Se ees wee alos ae eS Troubleshooting Guide 2 ee ee ee tee ene Ordering Information Introduction The E Z N A Tissue DNA Kit provides a rapid and easy method for the isolation of genomic DNA for consistent PCR and Southern analysis Up to 30 mg tissue or up to 1 cm sections of mouse tail can be readily processed in one time The method can also be used for preparation of genomic DNA from mouse tail snips blood buffy coat serum and plasma The kit allows single or multiple simultaneous processing of samples There is no need for phenol chloroform extractions and time consuming steps such as precipitation with isopropanol or ethanol are eliminated DNA purified using the E Z N A Tissue DNA method is ready for applications such as PCR Southern blotting and restriction digestion The E Z N A Tissue DNA Kit uses the reversible binding properties of HiBind matrix anew silica bas
8. ase 150 ul 1 4 ml 4x 1 4 ml User Manual 1 1 1 Buffer BL contains a chaotropic salt Use gloves and protective eyeware when handling this CAUTION solution 2 Before Starting IMPORTANT Wash Buffer must be diluted with absolute ethanol as follows D3396 00 Add 8 ml 96 100 ethanol D3396 01 Add 80 ml 96 100 ethanol per bottle D3396 02 Add 80 ml 96 100 ethanol per bottle Note All centrifugation steps must be performed at room temperature A E Z N A Protocol for Tissue Have the following reagents and supplies ready Tabletop microcentrifuge and sterile 1 5 ml tubes Warm up Elution Buffer 0 5 ml per sample to 70 C Have a shaking waterbath set to 55 C Absolute ethanol approximately 0 3 ml per sample RNase A Optional stock solution at 25 mg ml This method allows genomic DNA isolation from up to 30 mg tissue Yields vary depending on source OPTIONAL Although no mechanical homogenization of tissue is necessary pulverizing the samples in liquid nitrogen will improve lysis and reduce incubation time Once the liquid nitrogen has evaporated transfer the powdered tissue to a clean 1 5 ml tube Add 200 ul Buffer TL and proceed to step 2 below Mince up to 30 mg of tissue and place into a 1 5 ml microfuge tube Add 200 pl Buffer TL Cut the tissue into small pieces to speed up lysis For samples larger than 30 mg simply scale up the volume of Buffer TL used for a 60 mg sample use 400 ul buffer
9. d supplies ready Tabletop microcentrifuge and sterile 1 5 ml tubes Warm up Elution Buffer 0 5 ml per sample to 70 C Have a shaking waterbath set to 65 C Absolute ethanol approximately 0 3 ml per sample RNase A Optional stock solution at 25 mg ml This protocol is designed for rapid isolation of up to 25 ug genomic DNA from up to 5 x 10 cultured cells 1 Prepare the cell suspension 1a Frozen cell samples should be thawed before starting this protocol Pellet the cells by centrifugation wash the cells with PBS and resuspend cells with 200ul cold 4 C PBS Proceed with step 2 of this protocol 1b For cells grown in suspension pellet 5 x 10 cells by spinning at 1200 x g in a centrifuge tube Discard the supernatant and wash the cells once with PBS and resuspend cells with 200ul cold 4 C PBS 1c For cells grown in a monolayer harvest the cell by either using a trypsin treatment or scrape with rubber policemen Wash cells twice and resuspend the cells with 200ul cold 4 C PBS Add 25 ul of OB Protease and vortex to mix well Incubate at 65 C in a waterbath for 5 min to effect complete lysis OPTIONAL Cultured Cells have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point Add 5pl assuming 5 x 10 RNase A 25 mg ml and incubate at room temperature for 2 5 min Proceed with the tissue protocol Add 220 ul
10. e from Mollusc samples E Z N A Tissue DNA Kit please feel free to fax our technical specialists at D0926 01 02 Insect DNA Kit Isolation of total cellular DNA US customers 800 832 8896 a All other customers 770 931 8400 D4015 01 02 Stool DNA Kit Isolation of total cellular DNA Or direct your questions via E mail to info omegabiotek com from Stool samples 11
11. e quality of DNA can be assessed by measuring absorbance at both 260 nm and at 280 nm A ratio of A o A Of 1 7 1 9 corresponds to 85 95 purity Expected yields vary with both amount and type of tissue used 30 mg of fresh tissue will yield 10 40 pg DNA with two elutions each 200 yl Troubleshooting Guide Use the table below to find solutions to any problems you may have with the E Z N A Tissue DNA Kit Problem Possible Cause Suggestions Clogged Column Incomplete lysis Extend incubation time of lysis with Buffer TL and protease Add the correct volume of Buffer BL and incubate for specified time at 70 C It may be necessary to extend incubation time by 10 min Sample too large If using more than 30 mg tissue increase volumes of OB Protease or Proteinase K Buffer TL Buffer BL and ethanol Pass aliquots of lysate through one column successively Sample too viscous Divide sample into multiple tubes adjust volume to 250 ul with 10 mM Tris HCl Poor elution Repeat elution or increase elution volume see note on page 4 Incubation of column at 70 C for 5 min with Elution Buffer may increase yields Low DNA yield Wash Buffer Concentrate must be diluted with absolute 100 ethanol as specified on page 3 before use Improper washing Extended Resin from the column may be centrifugation present in eluate Avoid during elution centrifugation at speeds higher step
12. ed material combined with the speed of mini column spin technology A specifically formulated buffer system allows genomic DNA up to 60 kb to bind to the matrix Samples are first lysed under denaturing conditions and then applied to the HiBind spin columns to which DNA binds while cellular debris hemoglobin and other proteins are effectively washed away High quality DNA is finally eluted in sterile deionized water or low salt buffer Storage and Stability All components of the E Z N A Tissue DNA Kit can be stored at 22 C 25 C and are guaranteed for at least 24 months from the date of purchase Proteinase K should be stored at 15 C 25 C Under cool ambient conditions a precipitate may form in the Buffer BL In case of such an event heat the bottle at 37 C to dissolve Store Buffer BL at room temperature Binding Capacity Each HiBind DNA column can bind approximately 100 ug DNA Using greater than 30 mg tissue or 10 cells is not recommended The PCR process is covered by U S Patents 4 683 195 and 4 683 202 and international equivalents owned by Hoffmann LaRoche Inc Kit Contents Product D3396 00 D3396 01 D3396 02 Purification times 5 Preps 50 Preps 200 Preps l HiBind DNA columns 5 50 200 2 ml Collection Tubes 15 150 600 Buffer TL 3 ml 20 ml 60 ml Buffer BL 3 ml 20 ml 50 ml Buffer HB 3 ml 30 ml 110 ml DNA Wash Buffer 2 ml 20 ml 3 x 20 ml Elution Buffer 3 ml 30 ml 100 ml OB Prote
13. ial at this point If precipitation can be seen at this point break the precipitation by pipetting up and down 10 times Proceed step 7 14 of E Z N A Protocol for tissue on page 5 E Z N A Protocol for Paraffin Embedded Tissue Place not more than 30 mg tissue 2 mm in a clean 2 ml microfuge tube Extract the sample with 1 ml xylene to remove the paraffin Mix thoroughly by vortexing Centrifuge the tube at 10 000 x g for 10 min at room temperature Discard supernatant without disturbing the tissue pellet Rinse the pellet with 1 ml absolute ethanol to remove traces of xylene Centrifuge at 10 000 x g for 5 min at room temperature Discard the ethanol without disturbing the tissue pellet Repeat the ethanol rinse Air dry tissue pellet at 37 C for 15 min Add 200 ul Buffer TL to the tissue and follow E Z N A Protocol for Tissue from step 2 on page 4 Yields will depend on size and age of sample Certain samples may require prolonged lysis with Buffer TL Note Tissue fixed with paraformaldehyde will yield degraded DNA or RNA The extent of degradation depends on type of fixative used but the size of DNA obtained is usually less than 500 bp Degradation is not caused by the E Z N A Tissue DNA protocol and for PCR detection of segments smaller than 500 bp satisfactory results can be obtained E Vacuum Spin Protocol for Tissue DNA Extraction Carry out disruption homogenization Protease digestion and loading o
14. nto HiBind DNA column as indicated previous protocols Instead of continuing with centrifugation follow steps blow Note Please read through previous section of this book before using this protocol 1 Prepare the vacuum manifold according to manufacturer s instruction and connect the HiBind DNA V Spin column to the manifold 2 Load the sample into HiBind DNA V spin column 3 Switch on vacuum source to draw the sample through the column and turn off the vacuum 4 Wash the column by adding 500 ul Buffer HB draw the wash buffer through the column by turning on the vacuum source 5 Wash the column by adding 700 pl DNA wash buffer draw the wash buffer through the column by turning on the vacuum source 6 Wash the column again by adding 700 pl DNA wash buffer draw the wash buffer through the column by turning on the vacuum source 7 Assemble the column into a 2 ml collection tube and transfer the column to a micro centrifuge Spin at maxi speed no more than 20 000 x g for 2 minute to dry the column 8 Place the column in a clean 1 5 ml microcentrifuge tube and add 50 100pl DNA elution buffer Stand for 1 2 minute and centrifuge 1 minute to elute DNA Determination of Yield and Quality The total DNA yield can be determined by a spectrophotometer using deionized water Tris HCl buffer or Elution Buffer as blank DNA concentration is calculated as DNA Absorbance x 0 05 ug pl x Dilution factor Th
15. rmarked the animal return it to a clean cage Note Mice should not be older that 6 weeks since lysis will be more difficult resulting in suboptimal DNA yields If possible obtain tail biopsy at 2 4 weeks and freeze samples at 70 C until DNA is extracted Add 25 ul of OB protease and vortex to mix Incubate in a 55 C shaking waterbath for 1 4 hours or until lysis is complete If no shaking waterbath is available vortex vigorously every 20 30 min Incomplete lysis may block column flow and significantly reduce DNA yields Incubation time for complete tail lysis is dependent on length of tail and age of animal 0 5 cm tail pieces from 2 week old mice typically lyse in approximately 2 hours For older animals an overnight incubation may improve yields Note that bone and hair will not lyse Centrifuge for 5 min at 10 000 x g to pellet insoluble tissue debris and hair Carefully aspirate the supernatant and transfer to a sterile microfuge tube leaving behind any insoluble pellet OPTIONAL Mouse tail tissue contains RNA that can co purify with the DNA This will not interfere with PCR reactions but other enzymatic reactions may be affected To remove RNA add 15 ul of RNase A 25 mg ml and incubate 2 min at room temperature Add ONE volune of BL followed by ONE volume of ethanol Alternatively user can add 2 volume of premixed BL ethanol mixture to the sample Vortex thoroughly to mix at maxi speed for 15 sec Thoroughly mixing is essent
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