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1. Office 23 Vadhani Industrial Estate LBS Marg A 516 Swastik Disha Business Park Tel 00 91 22 6147 1919 ISO 9001 2008 ISO 13485 2003 Mumbai 400 086 India Via Vadhani Indl Est LBS Marg Fax 6147 1920 2500 5764 CERTIFIED CERTIFIED Tel 022 4017 9797 2500 1607 Mumbai 400 086 India Email info himedialabs com Fax 022 2500 2286 Web www himedialabs com The information contained herein is believed to be accurate and complete However no warranty or guarantee whatsoever is made or is to be implied with respect to such information or with respect to any product method or apparatus referred to herein cations and proteins are completely removed in two efficient wash steps leaving pure nucleic acid to be eluted in the buffer provided with the kit Elution Elution with 200 ul of Elution Buffer ET or Molecular Biology Grade Water will provide sufficient DNA to carry out multiple amplification reaction Elution with volume less than 200 ul will increase the final DNA concentration but will reduce the overall DNA yield Concentration yield and purity of DNA Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and the purity of genomic DNA Use Elution Buffer to dilute samples and to calibrate the spectrophotometer measure the absorbance at 260 nm 280 nm and 320 nm using a quartz microcuvette Absorbance readings at 260 nm should fall between 0 1 and 1 0 The 320 nm absorbance is us2. Cold Spring Harbor Sr No Problem Possible Cause Solution 1 Spin column is Lysate ethanol mixture is Vortex the tubes for atleast 5 10 seconds in clogged not homogenous order to obtain a homogenous solution before applying it to the column If minimally sheared DNA is required for downstream applications like PCR mix with gentle pipetting or inversion until homogenous lysate is obtained instead of vortexing DNA elution is improper Ensure that the DNA elution is in 200ul of Elution Buffer To improve the DNA yield incubate for 5 minutes at room temperature after Elution Buffer is added to the column Ethanol was omitted Ensure that ethanol is added in step 9 before during binding adding the sample to the spin column in step 10 Eluate contains residual Remove ethanol from the second wash ethanol from the wash completely before eluting the DNA Spin for an additional 2 minute to dry the membrane completely In order to avoid the interference of ethanol always use a fresh tube for elution Use of water instead of Elution Buffer is recommended for optimal Elution Buffer for elution yields and storage of the genomic DNA If of DNA water is used instead of the Elution Buffer the pH should be at least 7 0 to avoid acidic conditions which may cause acid hydrolysis of DNA when stored for long periods of time NOTE Only DNase RNase and Protease free water should be used for eluting DNA 2 Purity of the El
3. ble at room temperature 15 25 C upon reconstitution store at 20 C in dark wrapped in an aluminium foil as mentioned in storage instructions NOTE The 1M DTT solution must be added directly to each sample preparation every time 8 Reconstitute Proteinase K MBO86 The HiPurA Sperm Genomic DNA Purification Kit contains Proteinase K Intensive research has shown that it is the optimal enzyme for use with the Lysis Solution provided in the kit It is completely free of DNase and RNase activity Proteinase K is the enzyme of choice for use with an SDS containing Lysis Solution The specific activity of the Proteinase K is 33 5 units mg dry weight Resuspend the Proteinase K MB086 powder in Molecular Biology Grade Water MLO24 to obtain a 20 mg ml stock solution Number of Preps Proteinase K Molecular Biology Grade Water 20 10 mg 0 5 ml 50 24 mg 1 20 ml 250 120 mg 6 ml The product as supplied is stable at room temperature 15 25 C upon reconstitution store at 20 C as mentioned in storage instructions NOTE The Proteinase K solution must be added directly to each sample preparation every time Do not combine the Proteinase K and Lysis Solution for storage Ensure that clean amp dry tubes and tips are used for the procedure RNase A enzyme treatment RNase A is a type of RNase that is commonly used in research RNase A e g bovine pancreatic ribonuclease A is one of the sturdiest enzy
4. d collection tube NOTE Use a wide bore pipette tip to reduce shearing of the DNA while transferring contents into the column First Wash Prepare Wash Solution as indicated in General Preparation Instructions Add 700 ul of diluted Wash Solution WS to the column Centrifuge at 26 500 x g 10 000 rpm for 1 minute Discard the flow through liquid and re use the same collection tube with the column Second Wash Add another 700 ul of diluted Wash Solution WS to the column and centrifuge at 12 000 16 000 x g 13 000 16 000 rpm for 3 minutes to dry the column Discard the flow through liquid and centrifuge the column for another minute at the same speed if residual ethanol is observed NOTE i The column must be free of ethanol before eluting the DNA ii The collection tube can be emptied and re used for this additional centrifugation step 13 DNA Elution Place the column in a new 2 0 ml uncapped collection tube Pipette 100 ul of the Elution Buffer ET DSO040 directly onto the column without spilling to the sides Incubate for 1 minute at room temperature 15 25 C Centrifuge at 26 500 x g 10 000 rpm for 1 minute to elute the DNA Repeat the step again with another 100 ul of Elution Buffer ET for high yield of DNA NOTE DNA elution can also be performed in single step by the addition of 200 ul of Elution Buffer ET at a time DNA yield would be low Storing DNA in water may cause acid hydrolysis To increase the elut
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6. ed to correct for background absorbance An absorbance of 1 0 at 260 nm corresponds to approximately 50 ug ml of DNA The A260320 A2g0 A320 ratio should be 1 6 1 9 Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm DNA purified by HiPurA Sperm Genomic DNA Purification Kit is free of protein and other contaminants that can inhibit PCR or other enzymatic reactions Concentration of DNA sample ug ml 50 x Aso x dilution factor Materials needed but not provided Incubator at 55 C 70 C water bath or heating block Tabletop Microcentrifuge with rotor for 2 0 ml tubes Ethanol 96 100 Corex or any other suitable material centrifuge tubes Lymphocyte Separation Medium LSM Product Code LS001 Optional Clean and dry bottle of appropriate capacity for dilution of SEW Buffer Refer point 5 of General Preparation Instructions e Molecular Biology Grade Water Product code ML024 Storage Store the HiPurA Sperm Genomic DNA Purification Kit between 15 25 C except certain components as specified on each labels Under recommended condition kit is stable for 1 year General Preparation Instructions 1 Set the incubator at 55 C For incubation of semen sample semen pellet with Buffer SL to form semen lysate 2 Preheat a water bath or heating block to 70 C 3 Thoroughly mix reagents Examine the reagents for precipitation If any kit reagent forms a precipitate other t
7. f the sample Invert the tube intermittently for atleast 8 10 times for homogenous lysis of the sample during the incubation period If residual RNA is not a concern continue with step 8 to treat the semen lysate further NOTE More of intermittent shaking of the treated sample and less of pulse vortexing is recommended to prevent shearing of the sperm DNA NOTE If reduced incubation time is desired the user will have to optimize the incubation time at 55 C accordingly to ensure complete homogenization of the sample Optional RNase A treatment If RNA free genomic DNA is required add 20 ul of RNase A Solution DSO003 and incubate for 2 minutes at room temperature 15 25 C then continue with step 8 Lysis Add 200u of Lysis Solution C1 DS0010 to 200ul of semen lysate from step 7 Mix by vortexing thoroughly for 10 15 seconds A homogenous mixture is essential for efficient cell lysis Incubate at 70 C for 10 minutes NOTE If any clumps are visible the sample can be pipetted gently to obtain a homogenous mixture Prepare for Binding Add 200 ul of ethanol 96 100 to the lysate and mix thoroughly by vortexing for 5 10 seconds NOTE A homogenous solution is essential Load lysate in HiElute Miniprep Spin Column Capped DBCA03 Transfer the lysate obtained from step 9 onto the column provided Centrifuge at 26 500 x g 10 000 rpm for 1 minute Discard the flow through liquid and place the column in a same 2 0 ml uncappe
8. han enzymes warm at 55 65 C until the precipitate dissolves and allow cooling to room temperature 15 25 C before use 4 Preparation of Sperm Lysis Buffer SL DS0039 To 100 ul of Sperm Lysis Buffer SL add 20 ul of the Proteinase K solution 20 mg ml MBO86 Refer to step 8 of General Preparation Instructions and 8 ul of 1M DTT MBO70 just before use Mix thoroughly by vortexing for 10 15 seconds NOTE Sperm Lysis Buffer SL can be used effectively for semen as well as sperm sample 5 Dilute Semen Wash Buffer Concentrate SEW DS0038 as follows Number of Preps Semen Wash Buffer Concentrate SEW Molecular Biology Grade Water 20 12 ml 228 ml 50 28 ml 532 ml 250 135 ml 2565 ml NOTE User should carry out dilution of Semen Wash Buffer Concentrate SEW in a separate container as it is not provided with this kit 6 Dilute Wash Solution Concentrate WS DS0012 as follows Number of Preps Wash Solution Concentrate WS Ethanol 96 100 20 10 ml 30 ml 50 30 ml 90 ml 250 100 ml 300 ml 7 Reconstitute DTT DL Dithiothreitol MB070 Resuspend the DTT DL Dithiothreitol powder in Molecular Biology Grade Water Product code MLO24 to obtain a 1M stock solution Number of Preps DTT DL Dithiothreitol Molecular Biology Grade Water 20 30 85 mg 0 2 ml 50 77 12 mg 0 5 ml 250 385 62 mg 2 5 ml The product as supplied is sta
9. ie MolBi HIMEDIAT HIMEDIA Product Information Unzipping Genes MB522 HiPurA Sperm Genomic DNA Purification Kit Kit Contents Product Reagents provided plece Code 20 Preps 50 Preps 250 Preps ML116 Resuspension Solution 1X PBS 5ml 10 ml 50 ml DS0038 Semen Wash Buffer Concentrate SEW 12 ml 28 ml 135 ml DS0039 Sperm Lysis Buffer SL 4ml 8ml 30 ml DS0010 Lysis Solution C1 6ml 14 ml 65 ml DSO012 Wash Solution Concentrate WS 10 ml 30 ml 100 ml DS0040 Elution Buffer ET 10 mM Tris Cl pH 8 5 6 ml 13 ml 60 ml MBO86 Proteinase K 10 mg 24 mg 120 mg DS0003 RNase A Solution 20 mg ml 0 5 ml 1 1 ml 5 5 ml MB070 DL Dithiothreitol DTT 30 85 mg 77 12mg 385 62 mg a ee ee 22nos 58s 28000 DBCA016 Collection Tube Uncapped Polypropylene 2 0 ml 20 nos 50 nos 250 nos PW1139 Collection Tube Polypropylene 2 0 ml 40 nos 100 nos 2X 250 nos Introduction HiPurA Sperm Genomic DNA Purification Kit provides a fast and easy method for purification of total DNA for reliable applications in PCR and Southern blotting technique The DNA purification procedure using the miniprep spin column comprises of three steps viz adsorption of DNA to the membrane removal of residual contaminants and elution of pure genomic DNA HiMedia s HiElute Miniprep Spin Column Capped format allows rapid processing of multiple samples The columns have a high binding capaci
10. ion efficiency incubate for 5 minutes at room temperature 15 25 C after adding the Elution Buffer ET then centrifuge Elution with volume less than 200 ul increases the final DNA concentration in the eluate significantly but slightly reduces the overall DNA yield Storing DNA in water may cause acid hydrolysis 14 Transfer the eluate to a fresh capped 2ml collection tube for longer DNA storage Storage of the eluate with purified DNA The eluate contains pure genomic DNA For short term storage 24 48 hrs of the DNA 2 8 C is recommended For long term storage 20 C or lower temperature 80 C is recommended Avoid repeated freezing and thawing of the sample which may cause denaturing of DNA The Elution Buffer will help stabilize the DNA at these temperatures Alternative Short Protocol 1 Treatment with Sperm Lysis Buffer SL Prepare Sperm Lysis Buffer as indicated in General Preparation Instructions Add 100 ul of prepared Sperm Lysis Buffer SL DS0039 to 100 ul of semen sample Mix thoroughly by vortexing for 10 15 seconds Incubate at 55 C for 1 2 hours to ensure thorough lysis of the sample Invert the tube intermittently for atleast 8 10 times for homogenous lysis of the sample during the incubation period If residual RNA is not a concern continue with step 2 to treat the semen lysate further NOTE More of intermittent shaking of the treated sample and less of pulse vortexing is recommended to prevent shearing of
11. mes in common laboratory usage It cleaves 3 end of unpaired C and U residues Unit Definition for RNase A One unit of the enzyme causes an increase in absorbance of 1 0 at 260 nm when yeast RNA is hydrolyzed at 37 C and pH 5 0 Fifty units are approximately equivalent to 1 Kunitz unit It is completely free of DNases and proteases The specific activity is more than 90 U mg The product as supplied is stable at room temperature 15 25 C Centrifugation All centrifugation steps are carried out in conventional laboratory centrifuge e g Beckman CS 6KR Heraeus Varifuge 3 0R or Sigma 6k10 with fixed angle rotor The tubes provided with the kit are compatible with almost all laboratory centrifuges and rotors All centrifugation steps are performed at room temperature and are given in g the correct rpm can be calculated using the formula RPM VRCEF 1 118 x 10 r where RCF required gravitational acceleration relative centrifugal force in units of g r radius of the rotor in cm and RPM the number of revolutions per minute required to achieve the necessary g force Procedure 1 Washing of Semen Sample Add 10 ml of diluted Semen Wash Buffer SEW DS0038 Refer Step 5 of General Preparation Instructions to 100 200 ul of semen sample in a Corex or any other suitable material centrifuge tube and vortex at full speed for 10 seconds to ensure proper mixing NOTE Corex tubes are recommended as sperm cells do not adhere st
12. on buffer Safety Information The HiPurA Sperm Genomic DNA Purification Kit is for laboratory use only not for drug household or other uses The Lysis Solution C1 contains chaotropic salts which are irritant Take appropriate laboratory safety measures and wear gloves when handling Not compatible with disinfecting agents containing bleach Technical Assistance At HiMedia we pride ourselves on the quality and availability of our technical support For any kind of technical assistance send an email to mb himedialabs com PIMB522_0 0514 MB522 06 Disclaimer User must ensure of the product s in their application prior to use Products conform solely to the information contained in this and other related HiMedia Publications The information contained in this publication is based on our research and development work and is to the best of our knowledge true and accurate Himedia Laboratories Pvt Ltd reserves the right to make changes to specifications and information related to the products at any time Products are not intended for human or animal diagnostic or therapeutic use but for laboratory research orfurther manufacturing use only unless otherwise specified Statements contained herein should not be considered as a warranty of any kind expressed or implied and no liability is accepted for infringement of any patents d teal A 516 Swastik Disha Business Park Via Vadhani Indl Est LBS Marg Mumbai 400 086 In
13. rongly to the material of these tubes NOTE If only Sperm DNA is required the other cells present in the semen sample including lymphocytes should be separated out using appropriate gradient technique such as Lymphocyte Separation Medium LSM Product Code LS001 to obtain only sperms in the sample which will be treated further for extraction of DNA 2 Centrifuge at gt 2 500 x g 4 000 rpm for 10 minutes 3 Discard the supernatant leaving behind approximately 1 ml of pellet and Semen Wash Buffer SEW Vortex for 10 15 seconds to ensure proper mixing of pellet and diluted Semen Wash Buffer SEW and transfer the contents into a 2 0 ml capped collection tube 4 To the above sample add 0 5 ml of diluted Semen Wash Buffer SEW and vortex for 10 15 seconds to collect any sample adhering to the walls of the tube 5 Centrifuge at gt 6 500 x g 10 000 rpm for 2 minutes Carefully remove the supernatant avoiding removal of the semen pellet 6 Resuspension of pellet Resuspend the pellet in Resuspension Solution 1X PBS ML116 to make the final volume 100 ul 7 10 11 12 Treatment with Sperm Lysis Buffer SL DS0039 Prepare Sperm Lysis Buffer as indicated in General Preparation Instructions Add 100 ul of prepared Sperm Lysis Buffer SL DSO039 to 100 pl of resuspended semen pellet from Step 6 Mix thoroughly by vortexing for 10 15 seconds Incubate at 55 C for 1 2 hours to ensure thorough lysis o
14. the sperm DNA NOTE If reduced incubation time is desired the user will have to optimize the incubation time at 55 C accordingly to ensure complete homogenization of the sample Optional RNase A treatment NOTE RNase A treatment is generally not required while working with direct semen sample lysis method The RNase in the seminal plasma is generally sufficient to ensure degradation of the sperm RNA However if RNase treatment is still needed add 20 ul of RNase A Solution DSO003 to semen lysate from Step 1 and incubate for 2 minutes at room temperature 15 25 C Continue with step 2 2 Lysis Add 200u of Lysis Solution C1 DS0010 to 200uI of semen lysate from step 1 Mix by vortexing thoroughly for 15 seconds A homogenous mixture is essential for efficient cell lysis Incubate at 70 C for 10 minutes NOTE If any clumps are visible the sample can be pipetted gently to obtain a homogenous mixture 3 Continue with steps 9 13 of the regular procedure to extract sperm DNA from the sample Quality Control Each lot of HiMedia s HiPurA Sperm Genomic DNA Purification Kit is tested against predetermined specifications to ensure consistent product quality References 1 Sambrook J et al Molecular Cloning A laboratory Manual 2 Laboratory Press Plainview NY 1989 2 Birren B and Lai E Pulsed Field Gel Electrophoresis A practical guide Academic Press San Diego CA 1993 Troubleshooting guide d ed
15. ty and high quality genomic DNA is obtained from various species The DNA obtained is compatible with downstream applications such as restriction endonuclease digestion PCR and Southern blotting HiPurA Sperm Genomic DNA Purification Kit This kit simplifies isolation of DNA from sperm samples with spin column procedure Following lysis DNA binds to the silica gel membrane of the HiElute Miniprep Spin Column Capped to yield high purity DNA Two rapid wash steps remove trace salts and protein contaminants resulting in the elution of high quality DNA in the Elution Buffer ET provided with the kit HiElute Miniprep Spin Column Capped DBCA03 HiElute Miniprep Spin Column Capped is based on the advanced silica binding principle presented in a microspin format The system efficiently couples the reversible nucleic acid binding properties of the advanced silica gel membrane and the speed plus versatility of spin column technology to yield high quantity of DNA The use of spin column facilitates the binding washing and elution steps thus enabling multiple samples to be processed simultaneously This column eliminates the need for alcohol precipitation expensive resins and harmful organic compounds such as phenol and chloroform otherwise employed in traditional DNA isolation techniques DNA binds specifically to the advanced silica gel membrane while contaminants pass through PCR inhibitors such as divalent Registered Office Commercial
16. uate was diluted in Use either the Elution Buffer provided or 10 DNA is lower water for absorbance mM Tris HCl pH 8 5 than expected measurement Az60 Azgoratio is Background reading is Spin the DNA sample at maximum speed for 1 low high due to silica fines minute the supernatant can be used to repeat the absorbance readings 3 Purity of the RNA contamination RNase A treatment can be included in future DNA is higher isolations or the final product can be treated than expected with RNase A Solution and repurified Az60 Azgo ratio is Please refer disclaimer Overleaf too high Improper handling of genomic DNA All pipetting steps should be carried as gently as possible Wide bore pipette tips are recommended to eliminate shearing of the DNA to a large extent If the isolated DNA is to be used for PCR mix with gentle pipetting or invert until homogenous solution is obtained to reduce shearing of DNA considerably 4 Shearing of genomic DNA 5 Downstream applications are inhibited Traces of ethanol present in the final genomic DNA preparation After the washing steps the eluate should not come in contact with the column Spin the column for 1 minute at maximum speed 12 000 16 000 x g if necessary after emptying the collection tube Salt is carried over in the final genomic DNA preparation The spin column should be transferred to a new 2 0 ml collection tube before adding the eluti
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