INDEX KIT COMPONENTS 3 STORAGE AND STABILITY 3 BINDING CAPACITY 3 INTRODUCTION 3 IMPORTANT NOTES 4 EUROGOLD TISSUE DNA MINI KIT PROTOCOLS 5 A. DNA isolation from tissue 5 B. DNA isolation from eukaryotic cells (max. 5 x 10 6 cells) 6 C. DNA isolation from paraffin embedded tissue 7 D. DNA isolation from mouse tail snips 9 CONCENTRATING THE DNA 11 QUANTITATION AND STORAGE OF DNA 11 TROUBLESHOOTING 12
KIT COMPONENTS EuroGold Tissue DNA Mini Kits 5 Preparations 50 Preparations 200 Preparations Cat. EMR503005 EMR503050 EMR503200 Kit Components PerfectBind DNA Columns 5 50 200 2 ml Collection Tubes 15 150 600 DNA Lysis Buffer T 2 ml 24 ml 90 ml DNA Binding Buffer 1.5 ml 12 ml 45 ml DNA Wash Buffer 4 ml 40 ml 3 x 40 ml Elution Buffer (10 mm Tris-HCL, ph 9.0) 2 x 1.5 ml 25 ml 90 ml Proteinase K 3 mg 30 mg 120 mg RNase A (20 mg/ml) 75 µl 750 µl 2 x 1.5 ml 10 mm TE Buffer 1.5 ml 10 ml + 1.5 ml 30 ml + 4 x 1.5 ml Instruction manual 1 1 1 STORAGE AND STABILITY Dissolved Proteinase K should be stored at 20 C. RNase A should be stored at 4 C, all other components of the EuroGold Tissue DNA Mini Kits at room temperature. These components of the kits remain stable at an ambient temperature of 22 25 C for at least 12 months after the date of purchase. Precipitates can form in DNA Binding Buffer and should be dissolved by heating of the bottle at 37 C. BINDING CAPACITY Each PerfectBind DNA Column can bind approximately 30 µg of DNA. Using greater than 40 mg of tissue is not recommended. INTRODUCTION The EuroGold Tissue DNA Mini Kits provide a rapid and easy method for the isolation of up to 30 µg of genomic DNA from eucaryotic cells, fresh, frozen or paraffin-embedded tissue, dried blood or mouse tail snips. The kit allows simultaneous processing of single or multiple samples with up to 5 x 10 6 cells, 40 mg of tissue material or 0.8 cm of mouse tail snip. If dried blood should be processed we recommend the application of the EuroGold Blood DNA Mini Kits (EMR504XXX). There is no need for extractions with organic solvents like phenol or chloroform or time consuming steps such as precipitation with isopropyl alcohol or ethanol. Ed1/0709/EMR503/730 Page 3 of 13
Although the kits are principally also suited for the isolation of genomic DNA from blood, Buffy Coat, serum or plasma, we recommend the use of EuroGold Blood DNA Kits for these purposes. The EuroGold Blood DNA Kits are optimised for an efficient hemolysis and removal of hemoglobin and prove therefore better yields and higher quality of the isolated DNA. Genomic DNA purified with EuroGold Tissue DNA Mini Kits is ready for a wide range of applications such as PCR, Southern Blotting, and restriction digestion. EuroGold Tissue DNA Mini Kits use the reversible binding properties of the PerfectBind matrix, a silica-based material, combined with the speed of mini-column spin technology. A specifically formulated buffer system allows up to 30 µg of genomic DNA with fragment sizes up to 60 kbp to bind to the matrix. Samples are first homogenised, lysed under denaturing conditions and then applied to the PerfectBind DNA Columns, where the DNA is effectively bound to the silica membrane. Cellular debris, proteins and other contaminants are washed away by specific buffers. The high quality DNA is finally eluted in Elution Buffer or sterile deionized water. IMPORTANT NOTES Please read the protocol carefully before the first use of the EuroGold Tissue DNA Mini Kit and keep all required materials available before starting the preparation. DNA Binding Buffer contains a chaotropic salt. Use gloves and protective eyeware when handling this solution. Under low ambient temperatures, precipitates can form in DNA Binding Buffer. These precipitates are normal, but should be dissolved by heating the bottle at 37 C before use. DNA Wash Buffer is delivered as a concentrate and must be diluted with absolute ethanol before use: EMR503005 EMR503050 EMR503200 Mix 4 ml DNA Wash Buffer with 6 ml 100 % EtOH. Mix 40 ml DNA Wash Buffer with 60 ml 100 % EtOH. Mix 3 x 40 ml DNA Wash Buffer with 3 x 60 ml 100 % EtOH. Diluted DNA Wash Buffer should be stored at room temperature. If stored at lower temperatures, it should be heated to room temperature before use. Proteinase K is delivered as a powder and must be dissolved before use by thoroughly vortexing in TE Buffer: EMR503005 EMR503050 EMR503200 Dissolve 3 mg Proteinase K in 150 µl TE Buffer. Dissolve 30 mg Proteinase K in 1.5 ml TE Buffer. Dissolve 120 mg Proteinase K in 6 ml TE Buffer. Dissolved Proteinase K should be aliquoted in 25 µl, 50 µl or 100 µl, stored at 20 C and be freshly thawed before use. Ed1/0709/EMR503/730 Page 4 of 13
All centrifugation steps have to be performed at 22 25 C. EUROGOLD TISSUE DNA MINI KIT PROTOCOLS A. DNA isolation from tissue Required materials to be supplied by the user: 100 % ethanol Sterile pipette tips and centrifuge tubes 1. Homogenisation and Lysis Cut up to 40 mg of tissue (ca. 3 to 4 mm 3 ) in small pieces, place the sample in a 1.5 ml tube or mash the sample at the inside of the reaction tube with a pipette. Principally, a mechanical homogenization of the starting material is not required when isolating genomic DNA with the EuroGold Tissue DNA Mini Kit. Nevertheless, mechanical grinding under liquid nitrogen helps for a better lysis with shorter incubation times. Freeze the samples in a mortar and grind them with a pistil. After evaporation of the liquid nitrogen, take the sample up in 400 µl DNA Lysis Buffer T and continue the preparation as described in the following steps. Please note: liquid nitrogen can cause severe injuries. Always wear gloves and protective eyeware when using it. Add 400 µl DNA Lysis Buffer T, 20 µl of Proteinase K and 15 µl RNase A (20 mg/ml) and vortex for 10 sec. Incubate the sample at least at 50 C in a Thermo-Shaker. The incubation time depends on the type and amount of tissue to be prepared and is usually less than 3 hours. Lysis time can also be extended, but should not be continued, when the raw material is completely lysed. This could lead to degradation of the genomic DNA! If no Thermo-Shaker is available, mix the sample 3 to 4 times during incubation time by vortexing for 10 sec. Centrifuge the lysed solution for 30 sec at 10.000 x g to pellet down unlysable debris. Transfer the supernatant carefully in a new 1.5 ml tube without carrying-over parts of the debris pellet. 1) Loading and Binding Add 200 µl DNA Binding Buffer per 400 µl DNA Lysis Buffer T and mix thoroughly by pipetting. After addition of the DNA Binding Buffer a precipitate can form, which however has no influences on the DNA isolation. Place a PerfectBind DNA Column in a 2 ml Collection Tube and load up to 750 µl of the preparation (inclusive all precipitates) on the column. Centrifuge the PerfectBind DNA Column with the Collection Tube for 2 min at 10.000 x g. Discard the flowthrough. Repeat this step until the entire preparation is loaded. Discard the flowthrough and the Collection Tube. 2) Washing I Add 650 µl of the diluted DNA Wash Buffer (Buffer concentrate plus 1.5 volume absolute ethanol) on the column. Centrifuge the PerfectBind DNA Column with the Ed1/0709/EMR503/730 Page 5 of 13
Collection Tube for 1 min at 10.000 x g. Discard the flow-through, keep the Collection Tube. 3) Washing II Repeat the washing with 650 µl DNA Wash Buffer as described in step 3. Discard the Collection Tube. 4) Drying (Important step! Do not reduce centrifugation time!) Place the PerfectBind DNA Column in a Collection Tube and dry the column completely by centrifugation for 2 min at 10.000 x g. 5) Elution Place the PerfectBind DNA Column in a new 1.5 ml tube and pipette 200 µl Elution Buffer directly onto the membrane. Incubate for 3 min at room temperature, then centrifuge for 1 min at 6.000 x g. If necessary repeat the elution step once more with additional 200 µl of the Elution Buffer. The yields can be slightly improved by incubating the column at 70 C instead of room temperature before centrifuging or by using Elution Buffer pre-heated to 70 C. The final yield can be increased together with the concentration of the DNA, if the eluate is used for a second elution. But note that the yield is about 30 % decreased compared to a second elution with a fresh aliquot of Elution Buffer. Use a maximum of 40 mg of tissue per PerfectBind DNA Column to be prepared, otherwise the column will be overloaded. If you need to isolate higher amounts of DNA, divide the lysate on more than one PerfectBind DNA Column. Adapt the volume of buffers and solutions to bigger sample volume. For example add 400 µl DNA Lysis Buffer T and 40 µl Proteinase K solution to 80 mg of tissue material. Also adapt the volumes of DNA Binding Buffer in the following steps. B. DNA isolation from eukaryotic cells (max. 5 x 10 6 cells) Required materials to be supplied by the user: 100 % ethanol Sterile pipette tips and centrifuge tubes 1) Homogenisation and Lysis Suspension cells Pelletise cells by centrifugation (5 to 10 min at 5.000 x g). Discard the supernatant and resuspend the cells in 400 µl DNA Lysis Buffer T. Add 20 µl Proteinase K and 15 µl RNase A, mix thoroughly by vortexing for 10 sec and incubate at 50 C for 15 30 min in a Thermo-Shaker. If no Thermo-Shaker is available, mix the sample 3 to 4 times during incubation time by vortexing for 10 sec. Monolayer cells Cell culture cells growing in monolayers are directly lysed in the culture flask or dish. Discard the culture medium and add 400 µl DNA Lysis Buffer T, 20 µl Proteinase K solution and 15 µl RNase A per T35 flask or 10 cm dish. (For bigger samples adapt volumes of buffers and solutions.) Spread the buffer evenly over the whole surface to ensure complete detaching of the cells. Mix the lysate by pipetting up and down Ed1/0709/EMR503/730 Page 6 of 13
several times. Fill the lysate in a 5 ml centrifuge tube. Vortex for 10 sec and incubate at 50 C for 15 30 min in a shaking water bath. If no shaking water bath is available, mix the sample 3 to 4 times during incubation time by vortexing for 10 sec. 2) Loading and Binding Add 200 µl DNA Binding Buffer per 400 µl DNA Lysis Buffer T and mix thoroughly by pipetting. After addition of the DNA Binding Buffer a precipitate can form, which however has no influences on the DNA isolation. Place a PerfectBind DNA Column in a 2 ml Collection Tube and load up to 750 µl of the preparation (inclusive all precipitates) on the column. Centrifuge the PerfectBind DNA Column with the Collection Tube for 1 min at 10.000 x g. Discard the flowthrough. Repeat this step until the entire preparation is loaded. Discard the flowthrough and the Collection Tube. 3) Washing I Add 650 µl of the diluted DNA Wash Buffer (Buffer concentrate plus 1.5 volume absolute ethanol) on the column. Centrifuge the PerfectBind DNA Column with the Collection Tube for 1 min at 10.000 x g. Discard the flow-through, keep the Collection Tube. 4) Washing II Repeat the washing with 650 µl DNA Wash Buffer as described in step 3. Discard the Collection Tube. Place the PerfectBind DNA Column in a Collection Tube and dry the column completely by centrifugation for 2 min at 10.000 x g. 5) Elution Place the PerfectBind DNA Column in a new 1.5 ml tube and pipette 100-200 µl Elution Buffer directly onto the membrane. Incubate for 3 min at room temperature, then centrifuge for 1 min at 6.000 x g. If necessary repeat the elution step once more with additional 100-200 µl of the Elution Buffer. The yields can be slightly improved by incubating the column at 70 C instead of room temperature before centrifuging or by using Elution Buffer pre-heated to 70 C. The final yield can be increased together with the concentration of the DNA, if the eluate is used for a second elution. But note that the yield is about 30 % decreased compared to a second elution with a fresh aliquot of Elution Buffer. Use a maximum of 5 x 10 6 cells per PerfectBind DNA column for the preparation of genomic DNA, otherwise the PerfectBind DNA Column will be overloaded. If a higher number of cells needs to be processed, divide the lysate on more than one PerfectBind DNA Column. Adapt the volume of buffers and solutions on the bigger sample volume. C. DNA isolation from paraffin embedded tissue Required materials to be supplied by the user: Ed1/0709/EMR503/730 Page 7 of 13
Octane or xylene 100 % ethanol Sterile pipette tips and centrifuge tubes 1) Removal of paraffin Place up to 40 mg of tissue (ca. 3 to 4 mm 3 ) in a 2 ml centrifuge tube and remove the paraffin by vortexing thoroughly in 1 ml octane or xylene. The sample has to become transparent! Centrifuge for 5 min at maximum speed. Discard the whole supernatant carefully without destroying the tissue pellet. Repeat paraffin removal until paraffin is completely removed from the sample! 2) Washing of the tissue pellet Wash the tissue pellet with 1 ml of absolute ethanol by vortexing to remove the octane or xylene completely. Centrifuge for 3 min at maximum speed. Discard the supernatant containing ethanol without destroying the tissue pellet. Repeat this washing step once with another 1 ml of ethanol. Air-dry the tissue-pellet for 15 min at 37 C. 3) Lysis of the tissue pellet Add 400 µl DNA Lysis Buffer T and 25 µl Proteinase K solution to the tissue pellet and resuspend completely by vortexing for 10 sec. Incubate the preparation at 50 C in a Thermo-Shaker. The time required for the lysis depends on the type and amount of tissue to be prepared and is usually 30 min. Pre-heat thermomixer to 95 C during incubation time. The time required for the lysis of embedded material can be longer than for fresh tissues. If no Thermo-Shaker is available, mix the sample 3 to 4 times during incubation time by vortexing for 10 sec. Incubate sample in the thermomixer pre-heated to 95 C for 1 h. Do not place the lysate into the thermomixer until it has reached 95 C! 4) Loading and Binding Add 200 µl DNA Binding Buffer per 400 µl DNA Lysis Buffer T and mix thoroughly by pipetting. After addition of the DNA Binding Buffer a precipitate can form, which however has no influences on the DNA isolation. Place a PerfectBind DNA Column in a 2 ml Collection Tube and load up to 750 µl of the preparation (inclusive all precipitates) on the column. Centrifuge the PerfectBind DNA Column with the Collection Tube for 1 min at 10.000 x g. Discard the flowthrough. Repeat this step until the entire preparation is loaded. Discard the flowthrough and the Collection Tube. 5) Washing I Add 650 µl of the diluted DNA Wash Buffer (Buffer concentrate plus 1.5 volume absolute ethanol) on the column. Centrifuge the PerfectBind DNA Column with the Ed1/0709/EMR503/730 Page 8 of 13
Collection Tube for 1 min at 10.000 x g. Discard the flow-through, keep the Collection Tube. 6) Washing II Repeat the washing with 650 µl DNA Wash Buffer as described in step 5. Discard the Collection Tube. 7) Drying (Important step! Do not reduce centrifugation time!) Place the PerfectBind DNA Column in a Collection Tube and dry the column completely by centrifugation for 2 min at 10.000 x g. 8) Elution Place the PerfectBind DNA Column in a new 1.5 ml tube and pipette 50-100 µl Elution Buffer directly onto the membrane. Incubate for 3 min at room temperature, then centrifuge for 1 min at 6.000 x g. If necessary repeat the elution step once more with additional 50-100 µl of the Elution Buffer. The yields can be slightly improved by incubating the column at 70 C instead of room temperature before centrifuging or by using Elution Buffer pre-heated to 70 C. The final yield can be increased together with the concentration of the DNA, if the eluate is used for a second elution. But note that the yield is about 30 % decreased compared to a second elution with a fresh aliquot of Elution Buffer. Use a maximum of 40 mg of tissue per PerfectBind DNA Column to be prepared, otherwise the column will be overloaded. If you need to isolate higher amounts of DNA, divide the lysate on more than one PerfectBind DNA Column. Adapt the volume of buffers and solutions to bigger sample volume. For example add 800 µl DNA Lysis Buffer T and 40 µl Proteinase K solution to 80 mg of tissue material. Also adapt the volumes of DNA Binding Buffer in the following steps. The yield and the quality of the extracted DNA depends on the size and the age of the starting material. Tissue fixed with paraformaldehyd leads to degraded DNA and RNA. Although the degree of degradation of the nucleic acids depends on the fixative the resulting fragments are usually smaller than 500 bp. D. DNA isolation from mouse tail snips Required materials to be supplied by the user: 100 % ethanol Sterile pipette tips and centrifuge tubes 1) Homogenisation and Lysis Place 0.5 0.8 cm mouse tail or respectively 0.4 cm rat tail in a 1.5 ml tube and optionally in addition mash the sample at the inside of the reaction tube with a pipette tip. The mice should not be older than 6 weeks, otherwise the lysis will be difficult and the DNA yields will be decreased. The best results have been shown with samples from 2 to 4 week old mice. These samples can be stored at 70 C until the DNA isolation is done. The wound should professionally be treated and the mouse should be marked with an adequate label. Ed1/0709/EMR503/730 Page 9 of 13
Add 400 µl DNA Lysis Buffer T, 20 µl Proteinase K and 15 µl RNase A (20 mg/ml). Mix the lysate thoroughly by vortexing for 10 sec and incubate 2 to 3 h at 50 C in a Thermo-Shaker. Lysis time can also be extended, but should not be continued, when the raw material is completely lysed. If no Thermo-Shaker is available, mix the sample 3 to 4 times during incubation time by vortexing for 10 sec. Complete lyses of the mouse tail snips is not necessary for high yields and can lead to degradation of the genomic DNA. Centrifuge the lysed solution for 30 sec at 10.000 x g to pellet down unlysable debris. Transfer the supernatant carefully in a new 1.5 ml tube without carrying-over parts of the debris pellet. 2) Loading and Binding Add 400 µl DNA Binding Buffer per 400 µl DNA Lysis Buffer T and mix thoroughly by pipetting. After addition of the DNA Binding Buffer a precipitate can form, which however has no influences on the DNA isolation. Place a PerfectBind DNA Column in a 2 ml Collection Tube and load up to 750 µl of the preparation (inclusive all precipitates) on the column. Centrifuge the PerfectBind DNA Column with the Collection Tube for 1 min at 10.000 x g. Discard the flowthrough. Repeat this step until the entire preparation is loaded. Discard the flowthrough and the Collection Tube. 3) Washing I Add 650 µl of the diluted DNA Wash Buffer (Buffer concentrate plus 1.5 volume absolute ethanol) on the column. Centrifuge the PerfectBind DNA Column with the Collection Tube for 1 min at 10.000 x g. Discard the flow-through, keep the Collection Tube. 4) Washing II Repeat the washing with 650 µl DNA Wash Buffer as described in step 3. Discard the Collection Tube. 5) Drying (Important step! Do not reduce centrifugation time!) Place the PerfectBind DNA Column in a Collection Tube and dry the column completely by centrifugation for 2 min at 10.000 x g. 6) Elution Place the PerfectBind DNA Column in a new 1.5 ml tube and pipette 200 µl Elution Buffer directly onto the membrane. Incubate for 3 min at room temperature, then centrifuge for 1 min at 6.000 x g. If necessary repeat the elution step once more with additional 200 µl of the Elution Buffer. The yields can be slightly improved by incubating the column at 70 C instead of room temperature before centrifuging or by using Elution Buffer pre-heated to 70 C. The final yield can be increased together with the concentration of the DNA, if the eluate is used for a second elution. But note that the yield is about 30 % decreased compared to a second elution with a fresh aliquot of Elution Buffer. Ed1/0709/EMR503/730 Page 10 of 13
Do not use more than 0.8 cm of mouse tail snip per PerfectBind DNA Column for the preparation of genomic DNA, otherwise the column will be overloaded. If you need to isolate higher amounts of DNA, divide the lysate on more than one PerfectBind DNA Column. Adapt the volume of buffers and solutions to bigger sample volume. Use for example 800 µl of DNA Lysis Buffer T and 40 µl of Proteinase K solution for 1.6 cm mouse tail. Also adapt the volumes of DNA Binding Buffer in the following steps. CONCENTRATING THE DNA Genomic DNA purified with the EuroGold Tissue DNA Mini Kits can be further concentrated if required. Add NaCl to an end concentration of 0.1 M and 2 sample volumes of absolute ethanol. Incubate thoroughly by vortexing and incubate for 10 min at 20 C. Centrifuge for 15 min at 10.000 x g and discard the supernatant. Add 700 µl of 80 % ethanol and centrifuge for 2 min at 10.000 x g. Discard the supernatant, air-dry the pellet for 2 min and dissolve the DNA in 20 µl of Elution Buffer or sterile, deionised water. QUANTITATION AND STORAGE OF DNA To determine the concentration and the purity of a DNA containing solution, the absorbance of a 10- to 50-fold diluted aliquot is measured at 260 nm and 280 nm in a spectrophotometer. One A 260 unity corresponds to 50 µg of DNA per ml. The concentration can be determined as follows: DNA concentration (µg/ml) = absorbance 260 x 50 x dilution factor Alternatively the approximate yield and the quality of the received DNA can also be determined by agarose gel electrophoresis with subsequent ethidium bromide staining and comparison with wellknown DNA samples. The A 260/280 ratio of pure nucleic acids is 2.0. Generally genomic DNA isolated with the EuroGold Tissue DNA Mini Kit shows ratios between 1.7 and 1.9 corresponding to a purity of 85 % to 95 %. Genomic DNA isolated with the EuroGold Tissue DNA Mini Kits can be stored in Elution Buffer, 10 mm Tris-HCl (ph 9.0) or sterile, deionised water for several years at 20 C. Avoid frequent freezing and thawing as this will shear the DNA and result in a reduction of molecule size. Ed1/0709/EMR503/730 Page 11 of 13
TROUBLESHOOTING Problem Possible Cause Suggestions Clogged column Low DNA yield No DNA eluted Low A 260/280 ratio Problems with downstream applications Incomplete lysis Sample too large Sample too viscous Poor elution Improper washing Clogged column Poor cell and/or protein lysis in DNA Lysis Buffer T Poor cell lysis due to improper mixing with DNA Binding Buffer No ethanol added to DNA Wash Buffer concentrate Resin from the column present in the eluate Poor cell lysis due to incomplete mixing with DNA Binding Buffer Incomplete cell lysis or protein degradation Salt carry-over Ethanol carry-over Extend incubation time of lysis with DNA Lysis Buffer T and Proteinase K solution. Add the correct volume of DNA Binding Buffer and extend incubation time at 70 C by 10 min. Increase volumes of Proteinase K solution, DNA Lysis Buffer T and DNA Binding Buffer. Distribute lysate on more than one column. Dilute lysate with 10 mm Tris-HCl and divide sample into multiple tubes. Repeat elution or increase elution volume. Incubate column at 70 C for 5 min with Elution Buffer before centrifugation. DNA Wash Buffer concentrate must be diluted with ethanol before use. See above. Tissue sample must be cut or minced into small pieces. Increase incubation time at 50 C with DNA Lysis Buffer T. Mix thoroughly with DNA Binding Buffer prior to loading PerfectBind DNA Column. Dilute DNA Wash Buffer with the indicated volume of absolute ethanol before use. Avoid centrifugation at speeds higher than specified. The material can be removed from the eluate by centrifugation and will not interfere with enzymatic reactions. Vortex the sample with DNA Binding Buffer immediately and completely. Increase incubation time with DNA Lysis Buffer T and Proteinase K solution. Ensure that no visible pieces of tissue remain. Ensure that the DNA Wash Buffer is at room temperature. Dry the columns after the second washing for at least 2 min by centrifugation at 10.000 x g. Ed1/0709/EMR503/730 Page 12 of 13