Contents - Biomiga
Contents
1. Note Ensure that ethanol has been added to DNA Wash Buffer If refrigerated DNA Wash Buffer must be brought to room temperature before use 9 Using the same 2 mL collection tube Add 650 uL DNA Wash Buffer repeat Step 8 10 Using the same 2 mL collection tube centrifuge at a maximum speed 13 000 X g for 2 min to remove the residual ethanol Note This step is crucial for ensuring optimal elution 11 Place the ezBind DNA Mini Column into a sterile 1 5 mL microtube Add 100 200 uL preheated 70 C Elution Buffer Allow to sit at room temperature for 3 minutes Centrifuge at 13 000 x g for 1 min 12 Optional Repeat the elution with a second 100 200 uL Elution Buffer Note Each 200 pL elution will typically produces yields of 60 70 of the DNA bound to the column Thus two elutions will generally give 90 yields However increasing the elution volume will reduce the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 pL 100 pL of Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 pL greatly reduce yields In some instances yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer Page 10 Biomiga EZgene Tissue gDNA Miniprep Kit C EZgene Mouse Tail Genomic DNA Miniprep Protocol Bring frozen samples and Proteinase K solution to room temperature and p2. harvest the cell by scraping with a rubber policeman Wash cells twice and resuspend cells with 200 uL Buffer TL 2 Add 25 pL reconstituted Proteinase K Vortex to mix well and incubate in a 65 C shaking water bath to complete lysis effectively 3 OPTIONAL Cultured cells have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point To do so add 4 uL of RNase A 100 mg mL and incubate at room temperature for 2 minutes 4 Add 220 uL Buffer BL and vortex to mix Incubate at 70 C for 10 min Note A wispy precipitate may form upon addition of Buffer BL but does not interfere with DNA recovery 5 Add 220 pL Absolute Ethanol 96 100 room temperature and mix thoroughly by vortexing 6 Insert an ezBind DNA Mini Column into a 2 mL collection tube Transfer the entire sample from step 5 into the column including any precipitate that may have formed Centrifuge at 10 000 x g for 1 min to bind DNA Discard the collection tube with flow through liquid Page 9 Biomiga EZgene Tissue gDNA Miniprep Kit 7 Place the Mini Column into a new collection tube Add 500 pL Buffer KB Centrifuge at 10 000 x g for 1 min Discard collection tube and flow through liquid once again 8 Place the Mini Column into a new collection tube Add 650 uL DNA Wash Buffer diluted with absolute ethanol Centrifuge as above and discard flow through liquid
3. clean 1 5 mL tube Add 200 uL of Buffer TL and proceed to step 2 below 1 Mince up to 20 30 mg tissue and place it into a 1 5 mL microtube Add 200 uL Buffer TL In order to speed up lysis cut the tissue into small pieces For samples more than 30 mg simply scale up the volume of Buffer TL used For example 400 uL of Buffer TL should be used for 40 60 mg sample 2 Add 25 pL reconstituted Proteinase K Vortex to mix and incubate in a shaking water bath set to 55 C to effectively complete lysis If no shaking water bath is available vortex the sample every 20 30 min Note Lysis time depends on the amount and type of tissue used average time is usually under 3 hours One can allow lysis to proceed overnight 3 Optional Certain tissues such as liver tissue have high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point To do so add 4 uL assuming a sample size of 30 mg RNase A 20 mg mL and incubate at room temperature for 2 min Proceed with the tissue protocol 4 Centrifuge for 5 min at gt 13 000 x g to pellet insoluble tissue debris CAREFULLY aspirate the supernatant and transfer to a sterile microtube 5 Add 220 uL Buffer BL Vortex to mix Incubate at 70 C for 10 min Note A wispy precipitate may form upon addition of Buffer BL but does not interfere with DNA recovery Adjust the volume of Buffer BL required based on the amo
4. to sit at room temperature for 1 3 min Centrifuge at gt 13 000 x g for 1 min Optional Repeat the elution with a second 100 200 uL Elution Buffer Note Each 200 pL elution typically produces yields of 60 70 of the DNA bound to the column Thus two elutions will generally give 90 yields However increasing the elution volume will reduce the concentration of the final product To obtain DNA at higher concentrations elution can be carried out using 50 100 uL of Elution Buffer which slightly reduces overall DNA yield Volumes lower than 50 uL greatly reduce yields In some instances Page 7 Biomiga EZgene Tissue gDNA Miniprep Kit yields may be increased by incubating the column at 70 C rather than at room temperature upon the addition of Elution Buffer Page 8 Biomiga EZgene Tissue gDNA Miniprep Kit B EZgene Cultured Cell Genomic DNA Miniprep Protocol This protocol is designed for the rapid isolation of up to 25 ug of genomic DNA from up to 5 x 10 cultured cells 1 Prepare the cell suspension a Frozen cell samples should be thawed before starting this protocol Pellet the cells by centrifugation Wash the cells with PBS and resuspend cells with 200 uL Buffer TL b For cells grown in suspension pellet 5 x 10 cells by spinning at 1 200 x g in a centrifuge tube Discard the supernatant and wash the cells once with PBS Resuspend cells with 200 uL Buffer TL c For cells grown in a monolayer
5. Before Starting EZgene Kits are designed to be simple fast and reliable provided that all steps are followed diligently It is strongly advised that you familiarize yourself with the entire booklet before starting Important Notes MI Reconstitute each vial of Protease K 110 uL GD2211 00 1 3 mL GD2211 01 or 5 x 1 3 mL GD2211 02 of Elution Buffer Vortex the vial briefly before use We recommend that you aliquot and store vials of Proteinase K at 20 C Bring Proteinase K solution to room temperature before use MI Dilute DNA Wash Buffer with absolute ethanol as follows Add 8 mL GD2211 00 or 60 mL GD2211 01 or 96 mL GD2211 02 absolute ethanol 96 100 to each bottle MI Preheat Elution Buffer to 70 C in aliquots of 0 5 mL sample MI For Mouse Tail Protocol have the following ready Pre mixed solutions of 220 uL of Buffer BL and 220 uL of absolute ethanol per sample Mix by vortexing They can be prepared fresh or pre made and stored at room temperature less than one month Materials and Reagents to be supplied by user v Tabletop microcentrifuge M Sterile 1 5 mL centrifuge tubes V Shaking water bath set to 55 C for Tissue Protocol 65 C for Cultured Cells 55 C for Mouse Tail RNase Stock Solution 100 mg mL OPTIONAL Absolute Ethanol 96 100 PBS Buffer NWA RI Page 4 Biomiga EZgene Tissue gDNA Miniprep Kit Determination of Yield and Quality The total DNA yield can be de
6. Contents Introduction eie teri e pe nama 2 Storage and Stability ertet ette ette aan 2 Binding Capacity niei et et e ete e diete ces 2 Kat Contents A51 aset s ea oen e et een 3 Before Starting ic uei ue eU e RE e HER tee 3 EZgene Genomic DNA Miniprep Protocols A TAS SUG iso iere t ec ee nee tate eae eee 6 By Cultured Cell ete na Nissan 9 Ce Mouse Tails ento eno kun ss nana 11 D Paraffin embedded TissSue oooooWocaa 13 EZgene Vacuum Spin Protocol ooo oWoWooWoWW WWW 14 Trouble Shooting Guide eere 15 Related Products ec ewean mna 16 Page 1 Biomiga EZgene Tissue gDNA Miniprep Kit Introduction The EZgene family of products is an innovative system that radically simplifies the extraction and purification of nucleic acids from a variety of sources The key to this system is the new ezBind matrix that specifically but reversibly binds DNA or RNA under certain optimal conditions allowing proteins and other contaminants to be removed Nucleic acids are easily eluted with deionized water or a low salt buffer The EZgene Tissue DNA Kit provides an easy and rapid method for the isolation of genomic DNA for consistent PCR and Southern analysis Up to 30 mg of animal tissue culture cells mouse tail snips and paraffin embedded tissue can be readily processed at a time This kit allows for the single or multiple simultaneous processing of samples There
7. degradation depends on type of fixative used but the size of DNA obtained is usually less than 500 bp Degradation is not caused by the EZgene Tissue DNA Protocol For PCR detection of segments smaller than 500 bp satisfactory results can be obtained Page 13 Biomiga EZgene Tissue gDNA Miniprep Kit EZgene Vacuum Spin Protocol Carry out disruption homogenization protease digestion and loading onto the ezBind DNA Mini Column as indicated in previous protocols Instead of continuing with centrifugation follow the steps outlined below Note Please read through previous sections of this book before beginning this protocol 1 Prepare the vacuum manifold according to manufacturer s instruction and connect the ezBind DNA Spin Column to the manifold 2 Load the sample onto the column 3 Switch on vacuum to draw the sample through the column and then turn off the vacuum 4 Wash the column by adding 500 uL Buffer KB Draw the liquid through the column by turning on the vacuum 5 Wash the column by adding 650 uL DNA Wash Buffer Draw the Wash Buffer through the column by turning on the vacuum source 6 Washing the column with another 650 uL DNA Wash Buffer 7 Place the column into a 2 mL collection tube and transfer the column into a microcentrifuge Spin for 2 min at 13 000 x g with the lid open to dry the column 8 Place the column into a clean 1 5 mL microtube and add 100 200 uL of preheated 70 C E
8. ensuring optimal elution in the following step Place the Mini Column into a sterile 1 5 mL microtube and add 100 200 pL of preheated 70 C Elution Buffer Allow to sit at room temperature for 3 minutes Centrifuge at 13 000 x g for 1 min Optional Repeat the elution with a second 100 200 uL Elution Buffer Page 12 Biomiga EZgene Tissue gDNA Miniprep Kit D EZgene Paraffin Embedded Tissue Genomic DNA Miniprep Protocol 1 Place no more than 30 mg of tissue 2 mm in a clean 2 mL microtube 2 Extract the sample with 1 mL xylene not supplied to remove the paraffin Mix thoroughly by vortexing 3 Centrifuge the tube at 10 000 x g at room temperature for 10 min Discard the supernatant without disturbing the pellet 4 Rinse the pellet with 1 mL Absolute Ethanol to remove any traces of xylene Centrifuge at 10 000 x g for 5 min at room temperature Discard the ethanol without disturbing the tissue pellet 5 REPEAT the ethanol rinse 6 Air dry the tissue pellet at 37 C for 15 min 7 Add 200 uL Buffer TL to the tissue and then follow the standard Tissue DNA Isolation Protocol on page 6 starting with step 2 using reconstituted Proteinase K For DNA elution we recommend using 50 100 uL of Elution Buffer warmed to 70 C Yields will depend on size and age of sample Certain samples may require prolonged lysis with Buffer TL Tissue fixed with paraformaldehyde will yield degraded DNA or RNA The extent of
9. is no need for phenol chloroform extractions and time consuming steps are eliminated e g precipitation using isopropanol or ethanol Purified DNA can be directly used for most applications such as PCR Southern Blotting and Restriction Enzyme Digestion Storage and Stability All EZgene Tissue DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows reconstituted Proteinase K 20 C and all other materials at RT 22 25 C Binding Capacity Each ezBind DNA mini column can bind approximately 100 ug DNA Using greater than 30 mg tissue or 1x10 cells is not recommended Page 2 Biomiga EZgene Tissue gDNA Miniprep Kit Kit Contents Catalog GD2211 00 GD2211 01 GD2211 02 Preps 4 50 250 ezBind DNA Mini Columns 4 50 250 collection tube 8 100 500 Buffer TL 1 2 mL 15 mL 70 mL Buffer BL 1 2 mL 15 mL 70 mL Buffer KB 2 4 mL 28 mL 135 mL DNA Wash Buffer 2 mL 15 mL 3x24 mL Elution Buffer 2 mL 15 mL 70 mL Proteinase K 3 mg 30 mg 5 x 30 mg User Manual 1 1 1 Elution Buffer 10 mM Tris HCl pH 8 5 Add 8 mL GD2211 00 or 60 mL GD2211 01 or 96 mL GD2211 02 absolute ethanol 96 100 to each DNA Wash Buffer bottle before use Safety Information Buffer BL contains Chaotropic salts Use gloves and protective eyeware when handling this solution Page 3 Biomiga EZgene Tissue gDNA Miniprep Kit
10. ls and degraded 8 DNA Do not grow cell for longer than 16 hours Increase starting material and volume of all reagents Sample has low Proteinase K Buffer TL Buffer BL and ethanol DNA content proportionally Pass aliquots of lysate through column successively Resin from the column may be present in eluate Avoid Extended centrifugation at speeds higher than specified The material centrifugation VEN eae durine eluti can be removed from the eluate by centrifugation it will eee er not interfere with PCR or restriction digests Poor cell lysis due Repeat the procedure this time making sure to vortex the Low to incomplete sample with Buffer BL immediately and completely Asyl Aoso mixing with Buffer ratio 8 BL Incomplete cell lysis or protein degradation due to insufficient incubation Increase incubation time with Buffer TL and Proteinase K Ensure that no visible pieces of tissue remain Page 15 Biomiga EZgene Tissue gDNA Miniprep Kit Related EZgene Products Catalog Product Name Preps Price GD2211 02 250 420 00 mn YZ GD2311 01 Blood gDNA mini kit 90 00 GD2311 02 Blood gDNA mini kit 250 420 00 GD2312 01 Blood gDNA midi kit 10 9000 GD2312 02 Blood gDNA midi kit 25 20000 N ajo GD2314 01 Blood gDNA maxi kit 160 00 GD2314 02 Blood gDNA maxi kit 360 00 GD2411 02 250 495 00 GD2412 02 250 420 00 cA cA ajla GD2413 01 Insec
11. lution Buffer Let it sit at room temperature for 1 2 min and then centrifuge at 13 000 x g for 1 min to elute DNA 9 Place the Mini Column into a sterile 1 5 mL microtube and add 100 200 uL of preheated 70 C Elution Buffer Allow to sit at room temperature for 3 minutes Centrifuge at 13 000 x g for 1 min 10 Optional Repeat the elution with a second 100 200 uL Elution Buffer Page 14 Biomiga EZgene Tissue gDNA Miniprep Kit Trouble Shooting Guide Problem Possible reason Suggested improvement Extend incubation time of lysis with Buffer TL and iIneomelete Toa Proteinase K Add the correct volume of Buffer BL and P y incubate for specified time at 70 C It may be necessary to extend incubation time by 10 min e If using more than 30 mg tissue increase volumes of Sample is too Large Proteinase K Buffer TL Buffer BL and ethanol Pass aliquots of lysate through one column successively Sample is too Divide sample into multiple tubes and adjust the volume viscous to 25 uL with Buffer TL Clogged Column See above Repeat elution or increase elution volume see notes on Poor elution elution at the end of each protocol Incubation of column at 70 C for 5 min with Elution Buffer may increase yields enar wain DNA Wash Buffer must be diluted with absolute 96 Low DNA PIoP 8 100 ethanol as specified on page 4 before use Yield 1e Cultonsoversionn Overgrown culture contains lysed cel
12. re heat aliquots of Elution Buffer 0 5 mL sample at 70 C 1 Snip two pieces of mouse tail 0 2 0 5 cm in length Place into a sterile 1 5 mL microcentrifuge tube and add 180 uL Buffer TL Note If necessary cauterize the wound to stop bleeding Having appropriately earmarked the animal return it to a clean cage Mice should not be older than 6 weeks since lysis will be more difficult which results in suboptimal DNA yields If possible obtain a tail biopsy at 2 4 weeks and freeze samples at 70 C until DNA is extracted 2 Add 25 pL reconstituted Proteinase K Vortex to mix well and incubate in a shaking water bath set to 55 C for 1 4 hours or until lysis is complete If no shaking water bath is available vortex the sample every 20 30 min Note Incomplete lysis may block column flow and significantly reduce DNA yields Incubation time for complete tail lysis is dependent on tail length and animal age 0 5 cm tail pieces from a 2 week old mice typically lyses in approximately 2 hours For older animals an overnight incubation may improve yields Bone and hair will not be lysed 3 OPTIONAL mouse tail snips have low levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point To do so add 4 pL of RNase A 100 mg mL and incubate at room temperature for 2 minutes Proceed with the following protocol 4 Centrifuge for 5 min at 12 000 x g to pelle
13. t gDNA kit 90 00 GD2413 02 Insect gDNA kit 250 420 00 GD2415 01 Yeast gDNA kit 50 90 00 GD2415 02 Yeast gDNA kit 250 420 00 CA E ES GD2416 01 Fungal gDNA kit 90 00 GD2416 02 Fungal gDNA kit 250 420 00 Page 16 Biomiga EZgene Tissue gDNA Miniprep Kit
14. t insoluble tissue debris and hair CAREFULLY aspirate the supernatant and transfer to a sterile 1 5 mL microtube while leaving behind any insoluble pellet 5 Add 1 volume Buffer BL followed by 1 volume Absolute Ethanol Alternatively user may add 2 volumes of premixed BL ethanol mixture to the sample Vortex thoroughly to mix Thorough mixing is essential at this point Page 11 Biomiga EZgene Tissue gDNA Miniprep Kit 10 11 12 13 d DNA Mini Column into a 2 mL collection tube Transfer the entire sample from Step 5 into the column including any wispy precipitate Insert an ezBin that may have formed Centrifuge at 10 000 x g for 1 min to bind DNA Discard the collection tube with through liquid Place the Mini Column into a new collection tube Add 500 uL Buffer KB Centrifuge at 10 000 x g for 1 min Discard collection tube the flow through liquid Place the Mini Column into a new collection tube Add 650 uL DNA Wash Buffer Centrifuge at 10 000 x g for 1 min Discard flow through liquid Note Ensure that absolute ethanol has been added to the DNA Wash Buffer before use If refrigerated DNA Wash Buffer must be brought to room temperature before use Using the same 2 mL collection tube Add 650 uL DNA Wash Buffer repeat step 9 Using the same 2 mL collection tube centrifuge with the lid open at maximum speed of 13 000 x g for 2 min to remove residual ethanol Note This step is crucial for
15. termined by a spectrophotometer using deionized water Tris HCl Buffer or Elution Buffer as a blank DNA concentration is calculated as DNA Absorbance so x 0 05 ug uL x Dilution Factor The quality of DNA can be assessed by measuring absorbance at both 260 nm and 280 nm An A26 A280 ratio of 1 7 1 9 corresponds to 85 95 purity Expected yields vary with both amount and type of tissue used Typically 30 mg of fresh tissue will yield 10 40 ug of DNA with two elutions each 200 uL Concentrate DNA If necessary the DNA can be concentrated as follows Add sodium chloride to reach a final concentration of 0 1M followed by 2 x volume of absolute 96 100 ethanol Mix well and incubate at 20 C for 10 min Centrifuge at 10 000 x g for 15 min and discard supernatant Add 700 uL of 70 ethanol and centrifuge at 10 000 X g for 2 min Discard supernatant air dry the pellet for 2 min and resuspend the DNA in 20 uL of sterile deionized water or 10 mM Tris HCl pH 8 5 Page 5 Biomiga EZgene Tissue gDNA Miniprep Kit A EZgene Tissue Genomic DNA Miniprep Protocol This method is suitable for the isolation of genomic DNA from up to 30 mg of tissue Yields vary depending on source Optional Although no mechanical homogenization of tissue is necessary pulverizing the samples in liquid nitrogen will improve lysis and reduce incubation time Once the liquid nitrogen has evaporated transfer the powdered tissue into a
16. unt of starting material 6 Add 220 pL Absolute Ethanol 96 100 room temperature Vortex to mix Page 6 Biomiga EZgene Tissue gDNA Miniprep Kit 10 11 12 13 Note Adjust the volume of ethanol required based on the amount of starting material Insert an ezBind DNA Mini Column into a 2 mL collection tube Transfer the entire sample from step 6 into the column including any precipitate that may have formed Centrifuge at 10 000 x g for 1 min Discard the collection tube with flow through liquid Place the DNA Mini Column into a new 2 mL collection tube and add 500 uL Buffer KB Centrifuge at 10 000 x g for 1 min Discard collection tube and flow through liquid Place the DNA Mini Column into a new 2 mL collection tube Add 650 pL DNA Wash Buffer Centrifuge as above and discard flow through liquid Note Ensure that absolute ethanol has been added to the DNA Wash Buffer before use See label for directions If refrigerated DNA Wash Buffer must be brought to room temperature before use Using the same 2 mL collection tube Add 650 uL DNA Wash Buffer Repeat step 9 Using the same 2 mL collection tube and centrifuge with the lid open at maximum speed gt 13 000 x g for 2 min to remove the residual ethanol Note This step is crucial for ensuring optimal elution Place the DNA Mini Column into a sterile 1 5 mL microtube and add 100 200 uL preheated 70 C Elution Buffer 10 mM Tris pH8 5 Allow
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