User manual / Technical Information
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1. USER MANUAL AOTH Roti Prep Genomic DNA MINI For isolation of genomic DNA from tissues 36 gt J gt OER a Jong Buffer BSN r ee ee ae Dr w i m ae E Be 7 Ord No 8472 Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG Roti Prep Genomic DNA MINI For isolation of genomic DNA from tissues rodent tails FFPE tissues buccal swabs cell cultures Introduction and product description e Preparation by well known mini spin column system e Fast easy and efficient e Preparation in approx 15 minutes excluding lysis e Yield up to 65 ug gDNA depending on source material Roti Prep Genomic DNA MINI has been developed for the isolation of genomic DNA from various sources The isolation is carried out through the proven and reliable spin column technique which is very easy to handle and needs only few steps In addition to the especially on proteinase K adapted lysis buffer the filtration spin column has been optimised permitting particularly high extraction rates Extraction from up to 40 mg tissue therefore allows a yield of up to 65 ug gDNA The thereby extracted DNA is free of RNA and proteins and can directly be stored or used for downstream applications Figure Typical test gel following isolation of genomic DNA A and subsequent GAPDH PCR B from rodent tail frozen 8 mm each Yield of gDNA from left to right 22 6 ug 27 7 ug and 28 ug Ratio A260 A280 1 972. Mix by vortexing or pipetting until a homogenous solution is achieved 2 Column Loading Place each Spin Column into a 2 ml collection tube Apply the mix of sample Binding Buffer BSN to the spin column Centrifuge at 10 000 g or 12 000 rpm for 2 min RT and discard the flow through 3 Column Washing Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Again centrifuge columns at 12 000 14 000 rpm or full speed for 2 mins RT in order to remove residual ethanol 4 Elution Place the Spin Column into a clean 1 5 ml elution tube Add 200 ul Elution Buffer EB to the centre of the membrane Incubate for 1 min at room temperature Centrifuge at 6 000 g or 8 000 rpm for 1 min RT to elute DNA Alternatively adherent cells may be incubated in 96wells Wash cells with PBS and perform the lysis in the 96well under agitation Following lysis the cell solution has to be transferred into 1 5 ml tubes for centrifugation We recommend using a shaking platform or thermomixer for continuous agitation of the sample Alternatively vortex the sample 3 4 times during incubation Agitation is vital for a decent recovery rate If the solution has not comp
3. 0 01 Suitable source material e Tissue up to 40 mg e Rodent tails 0 5 1 cm e FFPE tissue paraffin embedded tissue e Buccal swabs e Cell culture up to 5x10 cells For research use only Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG Information in this document is subject to change without notice Carl Roth GmbH Co KG assumes no responsibility for any errors that may appear in this document Carl Roth GmbH Co KG disclaims all warranties with respect to this document expressed or implied including but not limited to those of merchantability or fitness for a particular purpose In no event shall Carl Roth GmbH Co KG be liable whether in contract tort warranty or under any statute or on any other basis for special incidental indirect punitive multiple or consequential damages in connection with or arising from this document including but not limited to the use thereof Cautions Lysis Buffer LSA Warning H319 P305 P351 P338 P337 P313 Binding Buffer BSN gt lt lt gt Danger H225 H318 H336 H411 P210 P280 P303 P361 P353 P305 P351 P338 P312 Proteinase K Danger H315 H319 H334 H317 H335 P260 P280 P342 P311 In case of forming of precipitates please dissolve by careful warming MSDS the appropriate MSDS can be downloaded from our website www carlroth com All due care and attention should be exercised in handling the materials and reagents contained in the kit Always wear gloves
4. g 100 ul Elution Buffer only increases the final concentration of DNA Repetition of the elution step often increases the yield of gDNA overall but may reduce the concentration after pooling of the fractions Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG 4 2 DNA isolation from paraffin embedded tissue samples Step RT room temperature done 1 Deparaffination Place the sample into a 2 0 ml tube add 1 ml Octane or Xylene Vortex carefully to dissolve the paraffin Monitor this process carefully until the tissue sample looks transparent while paraffin remains white Centrifuge at max speed for 5 mins RT Discard the supernatant very carefully by pipetting Do not remove the pellet Check the sample carefully and repeat the last step until the sample is completely transparent Add 1 ml ethanol 96 99 8 to the pellet and vortex vigorously Centrifuge at maximum speed for 3 min RT and remove the ethanol by pipetting Do not remove the pellet Add 1 ml ethanol 96 99 8 to the pellet and vortex vigorously Centrifuge at maximum speed for 3 min RT and remove the ethanol by pipetting Do not remove the pellet Incubate the open tube at 37 C for 10 15 mins to evaporate the residual ethanol 2 Tissue lysis Add 400 ul Lysis Buffer LSA 25 ul Proteinase K solution and 3 ul RNase A stock solution 100 mg ml not included in the kit Mix vigorously by pulsed vortexi
5. min RT to elute DNA We recommend using a shaking platform or thermomixer for continuous agitation of the sample Alternatively vortex the sample 3 4 times during incubation Agitation is vital for a decent recovery rate If the solution has not completely passed through the spin column centrifuge again at higher speed or prolong the centrifugation time Elution with lower volumes of Elution Buffer e g 100 ul Elution Buffer only increases the final concentration of DNA Repetition of the elution step often increases the yield of gDNA overall but may reduce the concentration after pooling of the fractions Carl Roth GmbH Co KG Roti Prep Genomic DNA MINI 4 4 DNA isolation from cultured cells 5 x 10 cells in maximum Adherent cells should be loosened from the culture bottle surface by trypsination or EDTA Remove cells and wash in PBS Count your cell number and select the appropriate cell amount 5x10 cells in maximum Step RT room temperature done 1 Cell lysis Pellet cells by centrifugation at 5 000 g or 7 500 rpm for 10 mins and discard the supernatant Add 200 ul Lysis Buffer LSA 25 ul Proteinase K solution and 3 ul RNase A stock solution 100 mg ml not included in the kit Mix vigorously by pulsed vortexing for 5 sec Incubate at 50 C under constant agitation until the sample is completely lysed approx 30 mins Add 200 ul Binding Buffer BSN to the sample
6. or 2 0 ml reaction tube and cut the shaft Add 400 ul Lysis Buffer LSA 25 ul Proteinase K solution and 3 ul RNase A stock solution 100 mg ml not included in the kit Mix vigorously by pulsed vortexing for 5 sec Incubate at 50 C under constant agitation for 10 15 mins Remove the swab by pressing the tip of the swab to the inner tube wall in order to quantitatively squeezing the liquid from the cotton wool into the tube Add 200 ul Binding Buffer BSN to the sample Mix by vortexing or pipetting in order to achieve a homogenous solution 2 Column Loading Place each Spin Column into a 2 ml collection tube Apply the mix of sample Binding Buffer BSN to the spin column Centrifuge at 10 000 g or 12 000 rpm for 2 min RT and discard the flow through 3 Column Washing Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Again centrifuge columns at 12 000 14 000 rpm or full speed for 2 mins RT in order to remove residual ethanol 4 Elution Place the Spin Column into a clean 1 5 ml elution tube Add 200 ul Elution Buffer EB to the centre of the membrane Incubate for 1 min at room temperature Centrifuge at 6 000 g or 8 000 rpm for 1
7. rpm for 1 min RT to elute DNA We recommend using a shaking platform or thermomixer for continuous agitation of the sample Alternatively vortex the sample 3 4 times during incubation Agitation is vital for a decent recovery rate Check lysis efficiency before proceeding in the protocol If the lysis is not complete prolong the incubation time in Lysis Buffer LSA This step is critical don t be impatient During lysis make sure to heat up a thermomixer or water bath to 90 C Only place the sample into the thermomixer when the temperature has reached 90 C If the solution has not completely passed through the spin column centrifuge again at higher speed prolong the centrifugation time Elution with lower volumes of Elution Buffer e g 100 ul Elution Buffer only increases the final or concentration of DNA Repetition of the elution step often increases the yield of gDNA overall but may reduce the concentration after pooling of the fractions Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG 4 3 DNA isolation from buccal swabs For a maximum yield of DNA the swab has to remain in the tube during the whole lysis procedure We recommend to cut the shaft of the swab in order to enable closing of the cap Removal of the swab from the tube ahead of time will lead to a dramatically reduced yield Step RT room temperature done 1 Tissue cell lysis Place the swab into a 1 5 ml
8. while handling these reagents and avoid any skin contact In case of contact flush eyes or skin with a large amount of water immediately Literature Citation When describing a procedure for publication using this product please refer to it as Carl Roth s Roti Prep Genomic DNA MINI Kit 1 Materials provided with the kit and storage conditions Amount Component Storage 6 30 5x30 mg Proteinase K 4 C 20 C 10 25 120 ml Lysis Buffer LSA gt 20 C 16 16 60 ml Binding Buffer BSN RT 6 24 2x60 ml Washing Buffer WST Base RT 2x2 15 2x30 ml Elution Buffer EB RT 10 50 250 Mini spin columns blue RT 20 100 500 1 5 ml Elution tubes RT 10 50 250 2 ml Collection tubes RT Carl Roth GmbH Co KG Roti Prep Genomic DNA MINI Lyophilized Proteinase K should be stored at 4 C Prior to use dissolve Proteinase K in 0 3 1 5 5x1 5 ml sterile distilled water as given below Dissolved Proteinase K should be stored in aliquots at 20 C Avoid repeated freeze amp thaw cycles for each tube Lysis Buffer should be stored at over 20 C in order to prevent precipitation of reagents The Roti Prep Genomic DNA MINI Kit should be stored dry at room temperature 14 25 C Before every use make sure that all components have room temperature If there are any precipitates within the provided solutions solve these precipitates by careful warming This kit is stable for at least 6
9. ent When elution DNA from buccal swabs we recommend to keep the swab in the tube during the whole lysis procedure in order to achieve a maximum yield of DNA In order to enable closing of the cap the shaft may be cut however Removal of the swab from the tube ahead of time will lead to a dramatically reduced yield Repetition of elution final step often enhances the recovery rate overall However even so often concentration of the recovered gDNA is reduced in parallel since the whole amount Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG of buffer used for elution is higher Elution is 100 ul Elution Buffer EB followed by another elution step in 100 ul Elution Buffer EB may be recommended Store the extracted DNA at 4 C For long time storage placing at 20 C is recommended For centrifugation we recommend a standard microcentrifuge Centrifugation steps should be carried out at room temperature Avoid repeated freeze amp thaw cycles for the tissue to be extracted Before start be sure to Add Ethanol 96 99 8 not incl in this kit to the Washing Buffer WST as follows 8472 1 10 Preps 14 ml 20 ml final vol 8472 2 50 Preps 56 ml 80 ml final vol 8472 3 250 Preps 140 ml to each bottle 2x 200 ml final vol Mix thoroughly and keep the bottle always firmly closed Dissolve Proteinase K by addition of distilled H20 as follows 8472 1 10 Preps 0 3 ml 8472 2 50 Preps 1 5 ml 8472 3 250 Pr
10. eps 1 5 ml to each tube Mix thoroughly Heat one thermal mixer or water bath to 50 C For elution from paraffin embedded tissue be prepared to heat another thermal mixer or water bath to 90 C Please note Centrifugation steps should be carried out at room temperature 4 Workflow The workflow has been provided on a separate page in order to use a copy for in process protocol or filing in your personal lab files Carl Roth GmbH Co KG Roti Prep Genomic DNA MINI 4 1 DNA isolation from tissue samples or rodent tails max 10 mg tissue or 1 cm tail section Step RT room temperature done 1 Tissue lysis Cut max 40 mg of tissue sample into small pieces and place the tissue into a 1 5 ml or 2 0 ml reaction tube Add 400 ul Lysis Buffer LSA 25 ul Proteinase K solution and 3 ul RNase A stock solution 100 mg ml not included in the kit Mix vigorously by pulsed vortexing for 5 sec Incubate at 50 C under constant agitation until the sample is completely lysed approx 0 5 2 h for tissue sample and 3 h for rodent tails After lysis has been completed centrifuge the lysed sample at 10 000 g or 12 000 rpm for 30 sec to spin down unlysed material Transfer the supernatant to a fresh 1 5 ml reaction tube Add 200 ul Binding Buffer BSN to the supernatant Mix by vortexing or pipetting until a homogenous solution is achieved 2 Column Loading Place each Spin Co
11. ionally prolong elution incubation time Degraded or sheared DNA Incorrect storage of starting material Make sure that the starting material is frozen immediately in liquid Nz or in minimum at 20 C and is stored continuously at 80 C Avoid repeated freezing and thawing of the starting material Old material Use fresh material Check and improve storage conditions RNA contamination Unsufficient RNAse digestion during lysis Add RNAse A during lysis Check storage conditions and usage of RNAse A Optionally replace RNAse A by a fresh lot 11 Carl Roth GmbH Co KG Roti Prep Genomic DNA MINI Your notes 12 Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG Ordering information for detailed kit content see Table under 1 ans Product Content Amount 8472 1 Roti Prep Genomic DNA MINI 1 Test Kit 10 Preps 8472 2 Roti Prep Genomic DNA MINI 1 Kit 50 Preps 8472 3 Roti Prep Genomic DNA MINI 1 Maxi Kit 250 Preps To place your order please contact us e Phone Germany 0800 56 99 000 e Phone international 49 0 721 5606 0 e Fax Germany and international 49 0 721 5606 149 e E Mail Germany bestellungen carlroth de e E Mail international info carlroth com 13
12. letely passed through the spin column centrifuge again at higher speed prolong the centrifugation time Elution with lower volumes of Elution Buffer e g 100 ul Elution Buffer only increases the final or concentration of DNA Repetition of the elution step often increases the yield of gDNA overall but may reduce the concentration after pooling of the fractions 10 Roti Prep Genomic DNA MINI Carl Roth GmbH Co KG 5 Trouble Shooting 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 material Check storage conditions and usage of Proteinase K Optionally replace Proteinase K by a fresh lot Reduce amount of starting material Overload of filters reduces yield Low recovery Insufficient lysis See above Insufficient mixing with Binding Buffer BSN Mix sample very well with Binding Buffer BSN by pipetting or by vortexing prior to transfer of the sample onto the Spin Filter Low amount of recovered gDNA Add the Elution Buffer directly onto the centre of the Spin Column Prolong the incubation time with Elution Buffer Increase volume of Elution Buffer used or repeat elution step Low concentration of eluted gDNA Reduce the volume of Elution Buffer used Opt
13. lumn into a 2 ml collection tube Apply the mix of supernatant Binding Buffer BSN to the spin column Centrifuge at 10 000 g or 12 000 rpm for 2 min RT and discard the flow through 3 Column Washing Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Again centrifuge columns at 12 000 14 000 rpm or full speed for 2 mins RT in order to remove residual ethanol 4 Elution Place the Spin Column into a clean 1 5 ml elution tube Add 200 ul Elution Buffer EB to the centre of the membrane Incubate for 1 min at room temperature Centrifuge at 6 000 g or 8 000 rpm for 1 min RT to elute DNA We recommend using a shaking platform or thermomixer for continuous agitation of the sample Alternatively vortex the sample 3 4 times during incubation Agitation is vital for a decent recovery rate Check lysis efficiency before proceeding in the protocol If the lysis is not complete prolong the incubation time in Lysis Buffer LSA This step is critical don t be impatient If the solution has not completely passed through the spin column centrifuge again at higher speed or prolong the centrifugation time Elution with lower volumes of Elution Buffer e
14. months after receipt when stored as directed Contents of this Kit may not be bought separately The user is responsible to validate the performance of the Roti Prep Genomic DNA MINI Kit for any particular use since the performance characteristics of our kits have not been validated for any specific application 2 Required Material and Equipment not included in this kit e Ethanol 96 99 8 e g Ethanol p a e g 9065 1 e Distilled sterilized H20 e RNAse A stock solution 100 mg ml e g 7156 1 100 mg solubilized in 1 ml distilled water made DNAse free by cooking e Xylene or octane optional 3 Application Roti Prep Genomic DNA MINI is designed for isolation of high purity genomic DNA from various source material Spin column based preparation allows elution in a small volume of low salt buffer eliminating time consuming phenol chloroform extraction or alcohol precipitation The columns may be used either in micro centrifuges or on vacuum manifolds The kit allows the extraction of up to 65 ug gDNA per preparation from up to 40 mg of rodent tails 0 5 1 cm paraffin embedded tissue buccal swabs or cultured cells up to 5x10 During lysis the sample has to be mixed carefully and if possible constantly Reduced movement of the lysis mix will reduce the lysis efficiency and subsequently the recovery rate of gDNA We recommend using a shaking platform or thermomixer in order to keep the sample at constant movem
15. ng for 5 sec Incubate at 50 C under constant agitation until the sample is completely lysed approx 0 5 2 h for tissue samples After lysis has been completed place the sample into a thermomixer prewarmed at 90 C and incubate the sample for 1 h Add 200 ul Binding Buffer BSN to the sample Mix by vortexing or pipetting until a homogenous solution is achieved 3 Column Loading Place each Spin Column into a 2 ml collection tube Apply the mix of sample Binding Buffer BSN to the spin column Centrifuge at 10 000 g or 12 000 rpm for 2 min RT and discard the flow through 4 Column Washing Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Add 700 ul of Washing Buffer WST to the Spin Column Centrifuge at 10 000 g or 12 000 rpm for 1 min RT and discard the flow through Again centrifuge columns at 12 000 14 000 rpm or full speed for 2 mins RT in order to remove residual ethanol Carl Roth GmbH Co KG Roti Prep Genomic DNA MINI Sequel DNA isolation from paraffin embedded tissue samples Step RT room temperature done 5 Elution Place the Spin Column into a clean 1 5 ml elution tube Add 200 ul Elution Buffer EB to the centre of the membrane Incubate for 1 min at room temperature Centrifuge at 6 000 g or 8 000
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