DNA Extraction November, 2011 (4th Edition)
Contents
1. M Solution CENDORI VeTeK nology Inc r must validate the system in compliance with the applicable law directives and or unlicensed nse to perform PCR or are not required to obtain a license2. 10 Add 700 ul of Washing Buffer to column and centrifuge at 13 000 rpm for 1 min Discard the flow through after centrifuging and place the MEGAquick spin column back in the same 2 ml collection tube Note If the DNA will be used for salt sensitive applications such as blunt end ligation and direct sequencing repeat the step 4 using 500 ul of Washing Buffer 11 Centrifuge for 1 min at 13 000 rpm to dry the spin membrane Note It is important to dry the spin membrane since residual ethanol may interfere with other reactions 12 Place the MEGAquick spin column to a clean 1 5 ml microcentrifuge tube not provided Apply 30 100 ul of the Elution Buffer directly to the center of the column without touching the membrane with the pipette tip Incubate at room temperature for 1 minute Centrifuge for 1 minute at 13 000 rpm 13 Discard the MEGAquick spin column and store the microcentrifuge tube containing the eluted DNA at 20 C Note It is suggested to use at least 30 ul of the Elution Buffer to obtain best result Note Ensure that the elution buffer is dispensed directly onto the MEGAquick spin membrane for complete elution of bound DNA The average eluate volume is 48 ul from 50 ul elution buffer volume and 28 ul from 30 ul Elution Buffer Note Elution efficiency is dependent on pH The maximum elution efficiency is achieved between pH 7 0 and 8 5 When using water make sure that the pH value is within this range and s
3. 1 min Recover fragment DNA Ss Ia 9 Com 9 Com ce 4 11 Additional Information SSS TROUBLESHOOTING GUIDE Problem Low or no yield DNA does not perform well e g in enzyme reaction ligation sequencing reactions BNL Buffer become violet color se A Bic Te gt m 2 27 Biotechnology Possible Cause Washing buffer did not contain ethanol Inappropriate elution buffer Incorrect volume of BNL buffer Gel slice incompletely solubilized Cloudy and gelatinous appearance of sample mixture after addition of isopropanol Salt concentration in eluate too high Eluate contaminated with agarose Eluate contains residual ethanol Eluate contain primer dimers pH of electrophoresis buffer too high Recommendation Ethanol must be added to Washing buffer before use DNA will only be eluted in low salt buffer or water Verify that a correct volume of BNL buffer was added to the gel slice After addition of BNL buffer to the slice mix by vortexing the tube every 2 minutes during the 55 C incubation This may be due to salt contamination and will be disappeared by mixing the sample Alternatively the gel slice may not be completely solubilzed The concentration of gel may be above 2 In this case apply the 6 volume of BNL buffer to gel slice and melt the gel completely Modify the washing step by incubating the column for 5min at RT after adding 70
4. Es tts 4 Effect of pH on DNA Binding The MEGAquick spin column is uniquely adapted to purify DNA from both aqueous solutions and agarose gels and up to 45 ug DNA can bind to each MEGAquick spin column The binding buffers in MEGAquick spin Total Fragment DNA Purification Kit provide the correct salt concentration and pH for adsorption of DNA to the MEGAquick spin column The adsorption of nucleic acids to silica surfaces occurs only in the presence of a high concentration of chaotropic salts which modify the structure of water Adsorption of DNA to silica also depends on pH Adsorption is typically 95 if the pH is 7 5 and is reduced drastically at higher pH Figure 1 If the loading mixture pH is gt 7 5 the optimal pH for DNA binding can be obtained by adding a small volume of 3 M sodium acetate pH 5 0 BNL Buffer contains an integrated pH indicator allowing easy determination of the optimal pH for DNA binding DNA adsorption requires a pH 7 5 and the pH indicator in the buffers will appear yellow in this range If the pH is gt 7 5 which can occur if during agarose gel electrophoresis the electrophoresis buffer had been used repeatedly or incorrectly prepared or if the buffer used in an enzymatic reaction is strongly basic and has a high buffering capacity the binding mixture turns orange or violet Fig 1 This means that the pH of the sample exceeds the buffering capacity of BNL Buffer and DNA adsorption will be ineffici
5. Other specialized analytical gel methods exist for analyzing extremely large or small DNA molecules Detailed information on all types of analytical gels can be found in current molecular biology manuals Agarose concentration The concentration of agarose used for the gel depends primarily on the size of the DNA fragments to be analyzed Low agarose concentrations are used to separate large DNA fragments while high agarose concentrations allow resolution of small DNA fragments Concentration of agarose used for separating DNA of different sizes Agarose conc DNA fragment Agarose conc DNA fragment Yo wiv range Kb Yo wiv range Kb 0 3 5 60 0 5 1 30 0 7 0 8 12 1 0 0 5 10 12 0 4 7 1 5 0 2 3 2 0 0 05 2 4 Electrophoresis buffers The most commonly used buffers for agarose gel electrophoresis are TBE Tris borate EDTA and TAE Tris acetate EDTA Although more frequently used TAE has a lower buffering capacity than TBE and is more easily exhausted during extended electrophoresis TBE gives better resolution and sharper bands and is particularly recommended for analyzing fragments 1 Kb The drawback of TBE is that the borate ions in the buffer form complexes with the cis diol groups of sugar monomers and polymers making it difficult to extract DNA fragments from TBE gels using traditional methods 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology 13 Additional Information
6. cut out the interesting DNA fragment with a sharp scalpel or razor blade Carefully take as much agarose gel as possible Note If sliced agarose gel put into BNL Buffer the total volume may be increased When highly concentrated BNL buffer is diluted and it results low elution efficiency Therefore minimize the size of the gel slice by removing extra agarose Note The gel slice may be stored at 4 C or 20 C for up to one week in a tightly closed tube under nuclease free conditions before purification g Weigh the gel slice in a 1 5 ml tube Add 3 volumes of BNL Buffer to 1 volume of gel 300 ul per 100 mg of agarose gel Note Add 300 ul of BNL Buffer to each 100 mg of gel If more than 2 of agarose gel add 6 volumes of BNL Buffer B Vortex the mixture and incubate at 55 C for 10 minutes or until the gel slice is completely dissolved To help dissolving gel vortex every 2 3 min during the incubation Note Vortex the tube every few minutes to increase the rate of agarose gel melting Centrifuge the tube briefly at room temperature to ensure that the contents are at the bottom of the tube Once the agarose gel is melted the gel will not resolidify at room temperature Note Completely solubilize agarose For gt 2 agarose gel increase incubation time Note If the color of the mixture is orange or violet add 10 ul of 3 M sodium acetate pH 5 0 and mix The color of the mixture will turn to yellow The adsorption of DNA to
7. isopropanol This step increases the yield of DNA fragment B Place one MEGAquick spin column in a Collection Tube for each DNA gel mixture san Transfer the DNA mixture to the MEGAquick spin column assembly D To bind DNA apply the sample to the MEGAquick spin column and centrifuge for 1 min Discard the flow through after centrifuging and place the MEGAquick spin column back in the same 2 ml collection tube Note The maximum volume of the MEGAquick spin column reservoir is 800 ul For sample volumes of more than 800 ul simply load and spin again N Add 700 ul of Washing Buffer to column and centrifuge at 13 000 rpm for 1 min Discard the flow through after centrifuging and place the MEGAquick spin column back in the same 2 ml collection tube Note If the DNA will be used for salt sensitive applications such as blunt end ligation and direct sequencing repeat the step 4 using 500 ul of Washing buffer so Centrifuge for 1 min at 13 000 rpm to dry the spin membrane Note It is important to dry the spin membrane since residual ethanol may interfere with other reactions 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology 10 Protocols Ea 9 Place the MEGAquick spin column to a clean 1 5 ml microcentrifuge tube not provided Apply 30 100 ul of the Elution Buffer directly to the center of the column without touching the membrane with the pipette tip Incubat
8. or TBE buffer Electrophoresis Sterile Absolute ethanol Standard tabletop micro centrifuge Micro centrifuge tubes sterile 1 5 ml TE buffer 10 mM Tris HCl 0 1 mM EDTA pH 8 0 8 5 2 INtRON MEGAquick spin Total Fragment DNA Purification Kit 27 Biotechnology Kit Information EE ui APPLICATIONS MEGAquick spin Total Fragment DNA Purification Kit is designed for the efficient isolation of DNA fragments from TAE or TBE agarose gels or direct purification of PCR products The purified DNA can be used for automated fluorescent DNA sequencing cloning restriction enzyme digestion and routinely performed DNA manipulation QUALITY CONTROL In accordance with iNtRON s ISO certified Total Quality Management System each lot of MEGAquick spin Total Fragment DNA Purification Kit is tested against predetermined specifications to ensure consistent product quality e The quality of the isolated fragment DNA was checked by agarose gel electrophoresis and spectrophotometric determination MEGAquick spin column control The DNA binding capacity was tested by determining the recovery obtained with 20 ug of input fragment DNA More than 7096 recovery was obtained e Buffer control Conductivity and pH of buffers were tested and found to be within the pre determinated ranges Buffer Conductivity BNL 190 200 mS cm Washing B 14 16 mS cm Elution 450 700 uS cm i INtRON MEGAquick spin Tot
9. reactions DNA yield and concentration DNA yield depends on the following three factors the volume of elution buffer how the buffer is applied to the column and the incubation time of the buffer on the column 100 200 ul of Elution Buffer completely covers the MEGAquick spin column ensuring maximum yield even when not applied directly to the center of the membrane Elution with 50 ul requires the buffer to be added directly to the center of the membrane and if elution is done with the minimum recommended volume of 30 yl an additional 1 minute incubation is required for optimal yield DNA will be up to 1 7 times more concentrated if the MEGAquick spin column is incubated for 1 minute with 30 ul of Elution Buffer than if it is eluted in 50 ul without incubation 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology 15 Additional Information EE 000 EXPERIMENTAL INFORMATION Yields of various sizes of Fragment DNA MEGAquick spin Total Fragment DNA Purification Kit shows improved DNA recovery from short size of DNA to long length fragment 20 Kb 87 bp 108 bp 218 bp 575 bp 1 0 kb 2 7 kb 4 5 kb 9 kb 20 kb MT Z3 1 2 3 1323 0 12 9 1313291 2 911 1313 3 4213 ww f j Fig 2 Yield of fragment DNA Fragment DNA Size 87 bp 20 Kb Lane M1 amp M2 DNA marker lane 1 Before purification l
10. the QlAquick membrane is efficient only at pH 7 5 BNL Buffer contains a pH indicator which is yellow at pH 7 5 and orange or violet at higher pH allowing easy determination of the optimal pH for DNA binding e Optional For 200 bp add 1 gel volume of isopropanol to dissolved gel solution of the step 5 and mix well by pipetting several times Do not centrifuge after mixing well Note For 200 bp of DNA fragment add 1 volume of isopropanol to 1 volume of BA gel and mix well If the agarose gel slice is 100 mg add 100 ul of isopropanol When adding the isopropanol and mixing well by pipetting small white pellet and clump should be formed But never mind and go to the following step This step increases the yield of DNA fragment For DNA fragment gt 200 bp adding isopropanol has no effect on yield 6 Place one MEGAquick spin column in a Collection Tube for each dissolved gel mixture 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology Protocols Ea 8 Transfer the dissolved gel mixture to the MEGAquick spin column assembly 9 To bind DNA apply the sample to the MEGAquick spin column and centrifuge for 1 min Discard the flow through after centrifuging and place the MEGAquick spin column back in the same 2 ml collection tube Note The maximum volume of the MEGAquick spin column reservoir is 800 ul For sample volumes of more than 800 ul simply load and spin again
11. 0 ul of Washing Buffer and the centrifuge If The gel slice is incompletely solubilized or over weighed increased the incubation time Ensure that the wash flow through is drained from the collection tube and that the column is then centrifuged at 13 000 rpm for 1min Primer dimers formed are longer than 50bp and are not completely removed After binding step wash the column with 750 ul of a 35 guanidine hydrochloride aqueous solution Follow with the washing and elution step as in the protocols The electrophoresis buffer has been repeatedly used or incorrectly prepared resulting in a sample pH that exceeds the buffering capacity of BNL Buffer and leads to inefficient DNA binding Add 0 1 volume of 3M sodium acetate pH 5 0 to the sample and mix MEGAquick spin Total Fragment DNA Purification Kit 12 Additional Information Es ll TECHNICAL ADVICE Principle of gel analysis Gels allow separation and identification of nucleic acids based on charge migration Migration of nucleic acid molecules in an electric field is determined by size and conformation allowing nucleic acids of different sizes to be separated However the relationship between the fragment size and rate of migration is non linear since larger fragments have greater frictional drag and are less efficient at migrating through the polymer Agarose gel analysis is the most commonly used method for analyzing DNA fragments between 0 1 and 25 Kb
12. 287 MEGAquick spin column Nucleic acid binding 200 Col Clear Column with column 17288 cap amp dark blue O ring 2 ml polypropylene Collection tube tube 50 tubes 200 tubes 1 BNL Buffer contains chaotropic salts which are irritants Take appropriate laboratory safety measures and wear gloves when handling 2 Washing Buffer is supplied as concentrate Add 40 ml 50 columns or 160 ml 200 columns per each bottles of ethanol 96 10096 according to the bottle label before use STORAGE MEGAquick spin Total Fragment DNA Purification Kit should be stored dry at room temperature 15 25 C Under these conditions MEGAquick spin Total Fragment DNA Purification Kit can be stored for up to 24 months without showing any reduction in performance and quality Check buffers for precipitate before use and redissolve at 37 C if necessary The entire kit can be stored at 2 8 C but in this case the buffers should be redissolved before use Make sure that all buffers and spin columns are at room temperature when used The term of validity is marked on the box INtRON MEGAquick spin Total Fragment DNA Purification Kit 27 Biotechnology Kit Information EE tie CONSIDERATION BEFORE USE typical agarose gel slice is solubilized by adding 3 volumes of BNL Buffer to 1 volume of gel e g 300 ul of BNL Buffer is added to 100 mg gel slice and incubating at 55 C for 10 minutes The high concentration of a ch
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14. DNA Extraction November 2011 4 Edition eel sel y vA Lr AP Segre Instruction manual MEGAquick spin M Total Fragment DNA Purification Kit The Instruction Manual for fragment DNA Extraction from agarose gel PCR product and enzymatic reaction using silica membrane 17286 I 17287 Ni 17288 NE 9 INtRON Biotechnology www intronbio com www intron innoplex com Copyright O 2011 iNTRON Biotechnology Inc All right reserved INDEX EE UA INDEX Kit Information Description 2 Consideration Before Use 4 Additonal Required Equipment Protocols Protocol A Gel DNA Extraction T Protocol B PCR Purification DNA Clean up ts Additional information Troubleshooting Guide 11 Experimental Information Se Wn ta INtRON MEGAquick spin Total Fragment DNA Purification Kit 49 Biotechnology Kit Information EE UA DESCRIPTION The MEGAquick spin Total Fragment DNA Purification Kit is designed to extract and purify DNA fragments of 60 bp 20 kb from normal or low melt agarose gels in either Tris acetate TAE or Tris borate TBE or to purify PCR products directly from a PCR amplification and DNA cleanup from other enzymatic reactions Recovery is achieved up to 95 PCR products are commonly purified to remove excess nucleotides and primers This membrane based system which can bind up to 45 ug DNA allows recovery of isolated DNA fragments or PCR products in as little as 20 minutes depending
15. Molecular diagnosis Phone 41 0 41 420 9636 Fax 41 0 41 420 9656 Molecular Diagnosis Austria URL http www lucerna chem ch Anopoli Biomedical Systems Iran Phone 43 2773 42564 Tunisia Sina Bio Medical Chemistry Co Fax 43 2773 44393 RIBO Pharmaceutique amp Phone 98 21 2244 2488 URL http www anopoli com Diagnostique Fax 98 21 2244 0888 Phone 216 71981095 URL http www sinabiomedical com Jordan Iraq Fax 216 71981473 Genetics Company Email ribopharma topnet tn Kazakhstan Phone 962 6 5536402 BioHim Pribor Fax 962 6 5536398 U S A Phone 7 727 278 23 16 URL http www genetics jo com Bulldog Bio Inc Fax 7 727 269 2791 Phone 1 603 570 4248 Email biohimpribor mail ru Malaysia Fax 1 603 766 0524 NHK BIOSCIENCE SOLUTIONS URL http www bulldog bio com Spain SDN EUROVET VETERINARIA S L Phone 60 3 7987 8218 Phone 34 91 8841374 Fax 60 3 7987 8213 Fax 34 918875465 URL URL http Avww euroveterinaria com http www nhkbioscience com Iran Mongolia Sina Bio Medical Chemistry Co SX Biotech Co Ltd Phone 98 21 2244 2488 Phone 976 5006 0677 Fax 98 21 2244 0888 Fax 976 7011 1767 URL http www sinabiomedical com Email zanaa sxbiotech mn 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology Do not hesitate to ask Us any question ea shop intronbio com 2600 1 Fox 82 505 550 5660 e Ma
16. R EELS 3 1 E EG E EE HE E EE I 1 1 1 0 B 1 COAG ACALAGCE GT CAACATATACACET CATE CCABACAGGAT CAAT CATA GT TARGET EC TECCOAAT ETE CECAAGGATAAG GAGE CAT OTS CEAAAGEEE EC NW ANA Hl ws NN M DO WM ON quum nm d HU 1 nin ni Unus OTT nn uiui unn nui n nui Hn 200709 tTeeceer or octet be TOCAACTOCCOCACA ACAGCOOCCOCAGCTETOATA 20 90024 ACARAAT b wama wamama AAA mr u Tm nieto u HIII usei TCCTCCOACT AGDAGCATTOCCECAACCASTEAGOCTOTECATEOOT essa TO6Ca077T0004404T0C4 Fig 5 Reliable Bons Read Lengths in Seieneing High quality sequencing data of 4 5 Kb length DNA fragment purified with iNtRON s MEGAquick spin Total Fragment DNA Purification Kit INtRON MEGAquick spin Total Fragment DNA Purification Kit gt O Biotechnology 17 Additional Information EE 0 0L 4 Molecular Reagent Australia Scientifix Pty Ltd Phone 61 3 85405900 Fax 61 3 9548 7177 URL http www scientifix com au Belgium European Biotech Network Phone 32 4 3884398 Fax 32 4 3884398 URL http www euro bio net com Canada FroggaBio Phone 1 416 736 8325 Fax 1 416 736 3399 URL http www froggabio com China Chinagen Inc Phone 86 0 755 26014525 Fax 86 0 755 26014527 URL http www chinagen com cn China Hong Kong Tech Dragon Limited Phone 852 2646 5368 Fax 852 2646 5037 URL http www techdragon com hk Egypt
17. al Fragment DNA Purification Kit 49 Biotechnology Kit Information EM COLUMN INFORMATION e The MEGAquick spin Total Fragment DNA Purification Kit Spin Column Column membrane Silica based membrane Spin Column Individually in inserted in a 2 0 ml Collection Tube Loading Volume Maximum 800 ul DNA Binding Capacity Maximum 45 pg Recovery 85 95 depending on the elution volume Elution Volume Generally eluted with 30 200 ul of Elution Buffer 1 Do not store the Column packs under completely dried conditions It may be affected to DNA binding capacity The Spin Columns are stable for over 2 year under these conditions TECHNICAL ASSISTANCE At iNtRON we are proud of ourselves on the quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of INtRON products If you have any questions or experience any difficulties regarding the MEGAquick spin Total Fragment DNA Purification Kit or INIRON products in general please do not hesitate to contact us OA INtRON MEGAquick spin Total Fragment DNA Purification Kit 49 Biotechnology Protocols EEE PROTOCOL A Gel DNA Extraction 1 Load and run the gel using an established protocol DNA can be extracted from standard or low melt agarose gels in TAE or TBE buffer po After electrophoresis
18. ane 2 after purification with older Product lane 3 after purification with MEGAquick spin Total Fragment DNA Purification Kit Comparative Test of DNA Recovery MEGAquick spin Total Fragment DNA Purification Kit shows the performance of competitive advantage of DNA extraction from all fragment size and amount oeo oet o vs O INIRON SupplierA e Supplier B A260 280 O INIRON E Supplier A E Supplier B Conc ng pl 108 218 9 9 1 300 2 700 4500 9000 20 000 Size of Fragment DNA bp Fig 3 Comparative performance test of fragment DNA purification Gel Ext Fragment DNA Size 108 bp 20 Kb INtRON MEGAquick spin Total Fragment DNA Purification Kit 49 Biotechnology 16 Additional Information iNRON e SupplierA e Supplier B o co x S lt OiNtRON E Supplier A E Supplier B Conc ng pl 300 500 Size of Fragment DNA bp Fig 4 Comparative performance test of DNA purification PCR purification Fragment DNA Size 1kb fragment linearized PCR fragment Start DNA amount 100 ng 2000 ng Average recovery MEGAquick spin Total 85 97 Supplier A 75 85 Supplier B 80 95 4 Suitable for Down stream Operations THH LAE noit uuu n ia ee AATY QT AATAC Ga C 1 red is AOOOCOAOCT Ce GTacccescace cones PRO AY KO JA HHH mnn mm EIU MATURE HIR PN ALERT pim NNNM KARI NA KINANA KANA EH B UEHEEEEEEE E B E EH L EL E EL 1 SEE
19. aotropic salt in BNL Buffer disrupts hydrogen bonding between sugars in the agarose polymer allowing solubilization of the gel slice In addition the high salt concentration dissociates DNA binding proteins from the DNA fragments f water is used for elution make sure that its pH is between 7 0 and 8 5 Elution efficiency is dependent on pH and the maximum elution efficiency is achieved within this range A pH 7 0 can decrease yield Note Store DNA at 20 C when eluted with water as DNA may degrade in the absence of a buffering agent SAFETY INFORMATION All chemicals should be considered as potentially hazardous When working with chemicals always wear a suitable lab coat and disposable glove Some buffers contain the chaotropic salt which may be an irritant and carcinogen so appropriate safety apparel such as gloves and eye protection should be worn If a spill of the buffers occurs clean with a suitable laboratory detergent and water If the liquid spill contains potentially infectious agents clean the affected area first with laboratory detergent and water then with a suitable laboratory disinfectant Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products 4 DO NOT add bleach or acidic solutions directly to the sample preparation waste ADDITIONAL REQUIRED EQUIPMENT Agarose iNtRON Cta No 32032 scalpel Gel running buffer TAE buffer
20. botag com Taiwan Asian Life Science Co Ltd Phone 886 2 2998 6239 Fax 886 2 8992 0985 URL http www asianscicom com tw Taiwan Hong jing Co Ltd Phone 886 2 3233 8585 Fax 886 2 3233 8686 URL http www hongjing com tw Thailand Pacific Science Co Ltd Phone 66 2 433 0068 Fax 66 2 434 2609 URL http www Pacificscience co th Turkey BIOCEM Ltd Co Phone 90 212 534 0103 Fax 90 212 631 2061 URL http www biocem com tr MEGAquick spin Total Fragment DNA Purification Kit 18 Additional Information EE 4 Molecular Reagent Kazakhstan Pakistan BioHim Pribor HR BIO SCIENCES adve Phone 7 727 278 23 16 Phone 92 42 37247650 b i i Fax 7 727 269 2791 Fax 92 42 37247650 CHEMBIO LTD Email biohimpribor mail ru E nai te uae eee au ou Phone 44 208 123 3116 hrbiosciences yahoo com Fax 44 800 007 3116 Spain mE Philippines URL http www chembio co uk EUROVET VETERINARIA S L fina Analytics INC Phone 34 91 8841374 Phone 632 461 7173 Boca Scientific URL il pldtdsl Phone 1 561 995 5017 Email hebborn g pldtdsl com Fax 1 561 995 5018 URL http www bocascientific com http www euroveterinaria com Romania S C Bio Zyme S R L Phone 40 264 52 32 81 vietnam Fax 40 264 52 32 81 PEU d URL http www biozyme ro Phone 844 37821739 m TES Fax 844 37821738 Switzerland Email info vietlab vn Qe Molecular Reagent CE
21. e at room temperature for 1 minute Centrifuge for 1 minute at 13 000 rpm 10 Discard the MEGAquick spin column and store the microcentrifuge tube containing the eluted DNA at 20 Note It is suggested to use at least 30 ul of the Elution Buffer to obtain best result Note Ensure that the elution buffer is dispensed directly onto the MEGAquick spin membrane for complete elution of bound DNA The average eluate volume is 48 ul from 50 ul elution buffer volume and 28 ul from 30 ul elution buffer Note Elution efficiency is dependent on pH The maximum elution efficiency is achieved between pH 7 0 and 8 5 When using water make sure that the pH value is within this range and store DNA at 20 C as DNA may degrade in the absence of a buffering agent The purified DNA can also be eluted in TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 0 but the EDTA may inhibit subsequent enzymatic reactions Guick Guide PCR Purification Gel Extraction DNA Clean up Transfer the lysates DNA samples U Excise the gel Weighing 13k rpm 1min 700 ul Washing Buffer FEN Transfer the sample 5 Vol of BNL Buffer SVoLoBNL Butter 3 Vol of BNL Buffer 13k rpm 1min For 200 bp add dbi is 1 Vol of isopropanol 1 Vol of isopropanol Column drying 13k rpm 1min Heating 50 pl Elution Buffer 3 N Assemble Columns 1 INtRON MEGAquick spin Total Fragment DNA Purification Kit 27 Biotechnology Centrifuge 13 000 rpm
22. ent In these cases the pH of the binding mixture can easily be corrected by addition of a small volume of 3 M sodium acetate pH 5 0 before proceeding with the protocol RN Optimal ph V V og pH High Reduced YA gt py i N z cs pH Fig 1 pH dependence of DNA adsorption MEGAquick spin column 1 ug of a 2 9 Kb DNA fragment was adsorbed at different pHs and eluted with Elution Buffer The graph shows the percentage of DNA recovery reflecting the relative adsorption efficiency versus pH of adsorption The pH indicator dye in MEGAquick spin Total Fragment DNA Purification Kit identifies optimal pH for DNA binding 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology 14 Additional Information E ia Low Salt Elution Elution efficiency is strongly dependent on the salt concentration and pH of the Elution Buffer Contrary to adsorption elution is the most efficient under basic conditions and low salt concentrations DNA is eluted with 50 or 30 ul of the provided Elution Buffer or water The maximum elution efficiency is achieved between pH 7 0 and 8 5 When using water to elute make sure that the pH is within this range In addition DNA must be stored at 20 C when eluted with water since DNA may degrade in the absence of a buffering agent Elution with TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 0 is possible but not recommended because EDTA may inhibit subsequent enzymatic
23. il intronbio intronbio com You can find your partners iNTRON Distributor in Page Multifunction Gel extraction PCR purification and DNA clean up Improved recovery Up to 9576 recovery of ready to use DNA Fast procedure Cleanup of DNA up 60 bp 20 Kb in three easy steps Prevention of error Easy determination of the optimal pH for DNA binding INNOPLEX GxN teleFAXgene CLP and IQeasy is a trademark of i tRON kits are intended for research use only Prior to using them for other pu regulations use of the PCR process Use of this product is recommended for persons that e 2011 iNtRON all rights reserved The PCR process is covered by patents issued and applicable in certain countri e WEST ZO NtRON Biotehc rposes the use ither have a lice ies iNtRON Biotechnology Inc does not encourage or support the unauthorized Rm 701 704 Jung Ang Induspia B D Sangdaewon dong Joongwon gu Sungnam si Kyeonggi do Korea iNtRON Biotechnology Inc Trademarks iNtRON DNA spin DNA midi DNA maxi PCRquick spin MEGA spin MEGAquick spin MEGA bead PROBER G DEX G spin Viral Gene spin easy spin RNA spin easy BLUE easy RED WEST on Broad Way PRO STAIN pLUG Maxime i Taq i StarTaq i MAX i StarMAX L gt PRO PREP SMART PRO MEASURE Genelator F Detector RedSafe Muta Direct e Myco
24. on the number of samples processed and the protocol used The purified DNA can be used for automated fluorescent DNA sequencing cloning labeling restriction enzyme digestion or in vitro transcription translation without further manipulation The BNL Buffer are optimized for efficient recovery of DNA and removal of contaminants As an added convenience from gel extraction procedures the BNL Buffer contains a color indicator that allows easy monitoring of the solution pH for optimal DNA binding e The MEGAguick spin Total Fragment DNA Purification Kit is ideal multi functional Gel extraction PCR purification and DNA clean up product for isolation of fragment DNA CHARACTERISTICS Multifunction Gel extraction PCR purification and DNA clean up Improved recovery Up to 95 recovery of ready to use DNA Simple and easy process Fast procedure Cleanup of DNA up 60 bp 20 Kb in three easy steps Prevention of error Easy determination of the optimal pH for DNA binding 2 INtRON MEGAquick spin Total Fragment DNA Purification Kit 27 Biotechnology Kit Information Es KIT CONTENTS Label Description ponent Contents 50 Columns 200 Columns Agarose gel lysis 1 BNL Buffer buffer 40 ml 140 ml Washing Buffer concentrate Washing buffer 10 ml 40 ml Elution Buffer Elution buffer 20 ml 20 ml ick In TM eae ap Nucleic acid binding 50 Col 200 Col Blue column w o Cap column 17296 17
25. tore DNA at 20 C as DNA may degrade in the absence of a buffering agent The purified DNA can also be eluted in TE buffer 10 mM Tris Cl 1 mM EDTA pH 8 0 but the EDTA may inhibit subsequent enzymatic reactions 5 INtRON MEGAquick spin Total Fragment DNA Purification Kit Biotechnology Protocols Essa PROTOCOL B PCR Purification DNA Clean up 1 Amplify target sample using standard amplification conditions or Prepare the DNA mixture enzymatically reacted for clean up N Add an 5 volume of BNL Buffer to the PCR reaction product and mix well by vortexing If the PCR product is 20 ul add 100 ul of BNL buffer to the PCR tube directly Note Centrifuge the tube briefly at room temperature to ensure that the contents are at the bottom of the tube Note If the color of the mixture is orange or violet add 10 ul of 3 M sodium acetate pH 5 0 and mix The color of the mixture will turn to yellow The adsorption of DNA to the MEGAquick spin membrane is efficient only at pH 7 5 BNL Buffer contains a pH indicator which is yellow at pH lt 7 5 and orange or violet at higher pH allowing easy determination of the optimal pH for DNA binding Ed Optional For lt 200 bp Add 1 5 volume of isopropanol to the sample and mix well by pipetting several times Do not centrifuge after mixing well Note For 200 bp Add 1 5 volume of isopropanol and mix well If the PCR product is 20 wl add 100 ul of BNL Buffer and 150 ul of
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