User Manual - XpressDNA Tissue Mini Kit
Contents
1. Buffer RNase A Solution Starting Material Use this procedure to isolate genomic DNA from 50 100 mg tissue Do not use more than 100 mg of tissue To obtain high yields of DNA and minimize DNA degradation use fresh samples or freeze the sample immediately in liquid nitrogen after collection Things to do before starting the protocol O Set the water bath or heat block at 56 C a Wash Buffer TI3 is provided as concentrate Before the first use add 96 100 ethanol as indicated on the bottle o Reconstitute the Proteinase K in Proteinase K Buffer volume mentioned on Proteinase K vial and store at 4 C after each use Procedure 1 Obtain the required tissue freshly from source or remove the tissue from storage If using frozen tissue equilibrate it to room temperature Cut and weigh 50 100 mg of tissue sample depends on the type of homogeniza tion After use store the remaining tissue according to the standard methods For ethanol preserved tissue take 50 100 mg of tissue and put it in a DNase free 1 5 ml microcentrifuge tube containing 500 ul of nuclease free water Incubate for 1 hour at room temperature to rehydrate the sample Then remove the water using a pipette and proceed to Step 2 for tissue May 2015 XpressDNA Tissue Mini Kit disruption and homogenization 2 Tissue disruption and homogenization can be performed by the following methods 2a Using a mortar and pestle in liquid nitrogen Place 50 100 m2. Carefully pipette the entire volume to avoid foaming as this might lead to sample loss Take care not to disturb the magnetic nanoparticles when on the MagnaStand The volume of nuclease free water used should be enough to resuspend the magnetic nanoparticles Incubate at 65 C for 5 minutes User Manual Problem DNA is sheared Cause Repeated freeze thaw of tissue samples Poor quality of tissue sample Contamination of work place and consumables with DNases Solution Avoid repeated freezing and thawing of tissue samples Do not use old tissue samples This often yields only degraded DNA Maintain a sterile environment while working Use DNase free consumables Inefficient performance of DNA in down stream applications Ethanol carryover Salt carryover Dry the separated magnetic nanoparticles containing the DNA to remove the ethanol after the wash steps Ensure that the correct amount of wash buffer is added and a total of two wash steps are per formed May 2015 XpressDNA Tissue Mini Kit APPENDIX Accessory Products Additional Products The table below lists additional products in pipeline available from MagGenome Technologies Pvt Ltd under the brand name XpressDNA for the isolation of genomic DNA from different sample types For more information about these and other XpressDNA Kits visit www maggenome com or contact Technical Support Product Quantity Ca
3. May 2015 XPRESS DNA Tissue Mini Kit CAT No 1404 TI 50 1404 TI 25 User Manual PN MagGenome TECHNOLOGIES PVT LTD www maggenome com XpressDNA Tissue Mini Kit Table of Contents Kit Contents 3 Shipping and Storage 3 Introduction 4 Before You Begin 5 General Informations 5 Sample Processing 5 Handling of MagNa Mix 5 MagnaStand 6 Elution 6 Safety Information 6 Protocol Extraction of Genomic DNA using XpressDNA Tissue Mini Kit 6 Troubleshooting Guide 10 Appendix 12 May 2015 2 User Manual Kit Contents The XpressDNA Tissue Mini Kit is available to process 25 and 50 samples XpressDNA Tissue Mini Kit XPD1404 TI 50 XPD1404 TI 25 Components Quantity XpressDNA Lysis Buffer 40 ml 20 ml XpressDNA MagNa Mix XpressDNA Wash Buffer 10 ml 5 ml Proteinase K Lyophilized 20 mg ml 10 mg 0 5 ml Proteinase K Buffer 1ml 0 5 ml RNase A Solution 20 mg ml 10 mg 0 5 ml Shipping and Storage All components of the XpressDNA Tissue Mini Kit are shipped at room temperature The RNase A Solution has to be stored at 4 C The lyophilized Proteinase K can be stored at room temperature for 6 months Before use reconstitute the Proteinase K with the buffer provided and store the reconstituted Proteinase K at 4 C upto 2 months For long term use make aliquots of reconstituted Proteinase K and store at 20 C 3 May 2015 XpressDNA Tissu
4. e Mini Kit Introduction The XpressDNA Tissue Mini Kit allows rapid and efficient purification of genomic DNA from both fresh and frozen tissues animal or human The genomic DNA is extracted using the XpressDNA magnetic nanoparticles based technology This approach facilitates easy inexpensive DNA isolations and avoid time consuming steps like centrifugation and column separation The extracted DNA is suitable for use in downstream applications including PCR restriction enzyme digestion etc The XpressDNA Technology The XpressDNA magnetic nanoparticles based technology works with a swappable charged solid surface which facilitates nucleic acid purification Under suitable buffering conditions the magnetic nanoparticles acquire a positive charge enabling the negatively charged DNA molecules to bind to them leaving the con taminants in the solution System Specifications Starting Material 50 100 mg tissue Elution Volume 100 ul DNA Yield 10 20 pg May 2015 4 User Manual Before You Begin It is important to read this section before starting the protocol to avoid non significant results General Information For the maximum recovery of the DNA go through the following information e Use consumables and equipments that are DNase free e Maintain a sterile environment to avoid DNase contamination e Perform recommended wash steps to avoid contaminants in the extracted DNA Sample Processing To obtain good results
5. from XpressDNA MagnaStand and add 100 ul of nuclease free water to the tube and gently pipette up and down 10 times to resuspend the magnetic nanoparticles Incubate the tube at 65 C for 5 minutes Place the tube in the XpressDNA MagnaStand for 2 minutes or until the solution appears clear With the tube on the XpressDNA MagnaStand carefully remove the supernatant containing the DNA to a sterile 1 5 ml microcentrifuge tube Take care not to disturb the separated magnetic nanoparticles Discard the used magnetic nanoparticles Store the purified DNA at 20 C or use immediately for the desired downstream application Avoid repeated freezing and thawing DNA Store the purified DNA at 4 C for short term use or aliquot the DNA and store at 20 C for long term storage May 2015 Troubleshooting Guide Problem Cause Incomplete lysis Poor quality of Low DNA yield tissue sample or No DNA recovered Improper handling of XpressDNA MagNa Mix Inefficient elution May 2015 XpressDNA Tissue Mini Kit Solution Start with lesser amount of tissue Do not use more than 100 mg of tissue Add appropriate amount of Proteinase K for lysis The tissue should be completely immersed in the lysis buffer Increase the incubation time at 56 C Properly store the tissue sample Process fresh tissues or frozen tissues that has been immediatly frozen after collection Vortex the MagNa Mix thoroughly before use
6. g the DNA use nuclease free water not supplied The volume of nuclease free water can be changed according to the required final concentration Be sure to use the nuclease free water volume not less than the volume required to resuspend the magnetic nanoparticles Safety Information Follow the safety guidelines below when using the XpressDNA Tissue Mini kit e Treat all reagents supplied in the kit as potential irritants e Always wear suitable lab coat disposable gloves and protective eye wears e Ifany spill occurs clean with a suitable laboratory detergent and water e If the liquid spill contains potentially infectious agents clean the affected area first with laboratory detergent and water then with 1 v v sodium hypochlorite or a suitable laboratory disinfectant Protocol Extraction of Genomic DNA using XpressDNA Tissue Mini Kit This protocol is designed for the isolation of genomic DNA from fresh frozen and ethanol preserved tissues Materials Required User Supplied e Tissue sample e Nuclease free water e Mortar amp pestle e XpressDNA MagnaStand or other magnetic separation rack e Sterile 1 5 ml microcentrifuge tubes May 2015 6 User Manual e Vortex mixer Sterile pipette tips 20 ul 200 wl and 1 ml e Water bath or heat block Components Supplied with the Kit e XpressDNA Lysis Buffer TI1 e XpressDNA MagNa Mix TI2 e XpressDNA Wash Buffer TI3 Proteinase K Lyophilized Proteinase K
7. g tissue in a mortar containing liquid nitrogen and grind the tissue using a pestle Transfer the tissue powder into a DNase free 1 5 ml microcentrifuge tube containing 750 ul Lysis Buffer Proceed to step 3 2b Using a mortar and pestle without liquid nitrogen Place 50 mg tissue in a mortar containing 250 ul Lysis buffer and grind the tissue using a pestle Transfer the tissue lysate into a DNase free 1 5 ml microcentrifuge tube containing 500 ul Lysis Buffer Proceed to step 3 2c Using a sterile blade Place 50 mg tissue in a sterile petri dish and cut the tissue into small pieces using a sterile surgical blade to increase the efficiency of tissue lysis Transfer the minced tissue into a DNase free 1 5 ml microcentrifuge tube containing 750 ul Lysis Buffer Proceed to step 3 3 Add 20 ul of RNase A to the lysate and mix by inverting the tube Incubate the tube at room temperature for 10 minutes 4 Add 20 ul of Proteinase K to the tube and mix by inverting the tube Note The incubation time after the addition of Proteinase K varies with the type of tissue homogenization 5 Incubate the sample according to steps 5a 5b 5c 5a Homogenization using motor and pestle with liquid nitrogen Incubate the sample for lysis at 56 C for 30 minutes or until the lysate appears clear 5b Homogenization using motor and pestle without liquid nitrogen Incubate the sample for lysis at 56 C for 45 minutes or until the lysate appears clear 5c Homo
8. genization using sterile blade Incubate the sample for lysis at 56 C until the lysate appears clear approximately 1 2 hours Note The lysis time varies with the type age and amount of tissue being used For soft tissues in 1 2 hours the lysis is completed and for fibrous tissue or rat tail an overnight incubation at 56 C is optimal 6 Add 350 ul of XpressDNA MagNa Mix to the digested tissue sample and May 2015 8 User Manual 10 11 12 13 14 15 16 17 18 mix by inverting the tube 5 times Incubate at room temperature for 5 minutes Note If using pipette use 1 ml pipette and set it to 900 ul to mix the sample Make sure that the entire volume is pipetted to avoid foaming as this may result in shearing of the DNA as well as sample loss Place the tube in the XpressDNA MagnaStand for 2 minutes or until the solution appears clear With the tube on the XpressDNA MagnaStand carefully remove and discard the supernatant Take care not to disturb the separated magnetic nanoparticles Add 500 ul of XpressDNA Wash Buffer to the tube and invert the XpressDNA MagnaStand up and down gently five times to wash the magnetic nanoparticles With the tube on the XpressDNA MagnaStand carefully remove the supernatant and discard Take care not to disturb the separated magnetic nanoparticles Repeat steps 9 10 for a total of two washes After completely drying the separated magnetic nanoparticles remove the tube
9. talogue No XpressDNA Blood Mini Kit 50 Reactions 1401 BL 50 25 Reactions 1401 BL 25 XpressDNA Bacteria Mini Kit 50 Reactions 1402 BA 50 25 Reactions 1402 BA 25 XpressDNA Fecal Mini Kit 50 Reactions 1406 FE 50 25 Reactions 1406 FE 25 Magna Stand 12 positions 1 XPD SD 12 May 2015 12
10. use fresh tissue or tissue that has been immediately frozen and stored at 20 C or 70 C Repeated freeze thaws of stored samples should be avoided since this leads to shearing of the DNA Use of poor quality starting material can also result in reduced yield and quality of the purified DNA The XpressDNA Tissue Mini Kit has been standardized using tissues like spleen liver kidney muscle and fish fin The kit is also optimized for the extraction of genomic DNA from ethanol preserved fish tissues Handling of MagNa Mix e Store the MagNa mix at room temperature e Before use mix the magnetic nanoparticles on vortex mixer e When added to the sample mix either by inverting the tube or by pipetting Try to avoid bubbles while mixing e Never dry the magnetic nanoparticles for too long after ethanol wash as it will reduce the efficiency of magnetic nanoparticles e Carefully remove the supernatant without disturbing or removing the magnetic nanoparticles e Do not freeze the magnetic nanoparticles as it will be damaged and cannot be used for DNA purification e Do not reuse the magnetic nanoparticles 5 May 2015 XpressDNA Tissue Mini Kit MagnaStand The MagnaStand consists of a base station and a tube holder molded together The tube holder can accomodate 12 microcentrifuge tubes The base of the MagnaStand contains six rare earth magnets that comes in contact with the microcentrifuge tube when in use Elution For elutin
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