AquaGenomic User Manual
Contents
1. AquaGenomic Solution is stable at room temperature 22 C However the micelles in AquaGenomic will settle down during storage and it is important that you vortex it vigorously for 30 60 minutes to mix the solution well before dispensing 2 Should I keep the solutions and samples on ice while carrying out the isolation No Most of the isolation steps should be performed at room temperature 22 C For DNA isolation from animal cells cell lysis at 60 C may increase the yield by 25 For DNA isolation from cells with a cell wall such as bacterial cells the cell lysis should be carried out at 60 90 C for 15 30 minutes 3 Does AquaGenomic Solution contain Proteinase K No AquaGenomic can be used to extract DNA from cells and tissues without needing protease digestion However if mitochondrial DNA mtDNA recovery is desired you will need to add Proteinase K to AquaGenomic solution to 100 ug ml and incubate the sample at 60 65 C for 1 hour during cell lysis and then at 95 for 10 minutes to inactivate the Proteinase K 4 What type of homogenizer do you recommend for using with AquaGenomic Pestle and tube homogenizers produce more homogeneous samples However if you do not have a homogenizer handy or you are concerned about cross contamination you could use the Proteinase K digestion protocol see AquaGenomic Tissue Protocol for details to extract the DNA 5 Does AquaGenomic Solution contain RNase A No AquaGenomic S2. for better yield for 4 minutes 3 Pellet the Debris Optional Add 5 ul 100 isopropanol to the sample and vortex to reduce foaming and allow the debris pellet become tighter following centrifugation Vortex vigorously for 30 60 seconds and centrifuge the sample at 10 000 20 000xg for 2 minutes to pellet the debris 4 Pellet the DNA Transfer the supernatant 90 ul to a fresh 0 5 ml microfuge tube Add 1 volume of 100 isopropanol and mix the contents by vortexing for 30 seconds Centrifuge at 10 000 20 000xg for 4 minutes to pellet the DNA Flip to discard the supernatant Fill the microfuge tube with 70 ethanol by shooting the ethanol solution at the cap of the tube from a squeeze bottle and then flip to discard the ethanol Repeat the 70 ethanol rinse 2 times Flip the tube and blot it several times on a paper towel to remove residual ethanol Air dry the DNA pellet Add 50 ul of TE buffer or deionized water pipette up and down and vortex vigorously to suspend the DNA Centrifuge again for 2 minutes to pellet any insoluble and transfer the clear DNA solution to a new tube Store the DNA solution at 4 or at 20 C for long term storage MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Tissue Protocol DNA may be extracted from tissues by homogeniz
3. AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Instruction Manual General Information Description AquaGenomic is a multifunctional aqueous solution for genomic DNA isolation and purification This single solution functions to lyse cells extract the DNA and precipitate cell debris It can be used to isolate genomic DNA from bacteria to animal tissues The protocols are simple fast and scalable Approximately 5 10 ug DNA can be isolated from 1 2 million cultured cells 1 ml of bacterial cultures 5 10 mg of animal tissues or 10 20 mg of plant tissues A260 A280 of the isolated DNA is 1 6 1 8 Specification Product Name AquaGenomic Kit Product 2001 2030 Size 2001 For 10 minipreps from cultured cells 2030 For 300 mini 30 midi and 3 maxi preps from cultured cells Kit Contents 2001 1 ml AquaGenomic Solution Instruction Sheet 2030 30 ml AquaGenomic Solution Instruction Manual MSDS Available at www aquaplasmid com Storage Store tightly capped at RT 22 C Vortex to mix well before using Terms amp Condition Product Usage For In Vitro Laboratory Research Use Only NOT to be administered to humans or used for medical diagnosis Limited Product Warranty We offer a LIMITED PRODUCT WARRANTY to our customers This warranty limits our liability to replacement of this product No other warranties of any kind express or implied including without limitation implied wa
4. Proteinase K e g add 2 ul of 5 mg ml Proteinase K stock solution to 100 ul of AquaGenomic solution just before the extraction 2 Extract the DNA Incubate at 65 C for 90 minutes and then at 95 C for 10 minutes to inactivate the Proteinase K The tissue is readily disintegrated by vortexing vigorously or pipetting 3 Pellet the Debris Centrifuge at 12 000 20 000xg for 4 minutes to pellet the debris 4 Pellet the DNA Transfer the supernatant 80 ul to a fresh microfuge tube Add 1 volume of 100 isopropanol and mix the contents by vortexing for 30 seconds Centrifuge at 10 000 20 000xg for 4 minutes to pellet the DNA Flip to discard the supernatant Fill the microfuge tube with 70 ethanol by shooting the ethanol solution at the cap of the tube from a squeeze bottle and then flip to discard the ethanol Repeat the 70 ethanol rinse 2 times Flip the tube and blot it several times on a paper towel to remove residual ethanol Air dry the DNA pellet Add 50 ul of TE buffer or deionized water pipette up and down and vortex vigorously to suspend the DNA Centrifuge again for 2 minutes to pellet any insoluble and transfer the clear DNA solution to a new tube Store the DNA solution at 4 or at 20 C for long term storage MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation sol
5. ex vigorously to suspend the DNA Centrifuge again for 2 minutes to pellet any insoluble and transfer the clear DNA solution to a new tube Store the DNA solution at 4 or at 20 C for long term storage MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Microbe Protocol This protocol can be used to prepare 10 20 ug of genomic DNA from 1 ml overnight microbial culture For other preparation scales use 100 ul of AquaGenomic Solution for each milliliter of overnight culture IMPORTANT The micelles in AquaGenomic solution may fuse and settle down during storage so vortex vigorously to mix well before dispensing 1 Harvest the Cells Centrifuge 1 ml overnight bacterial culture at 10 000 20 000xg for 30 seconds to pellet the cells Aspirate to discard the medium and leave behind the cell pellet 2 Extract the DNA For Gram negative bacteria Add 100 ul of AquaGenomic Solution to the cell pellet Suspend the cells by vortexing vigorously for 30 seconds Incubate the sample at 65 C for 15 minutes to lyse the cells For some strains cell lysis and DNA yield may be enhanced by incubating at 90 C for 30 minutes For Gram positive bacteria or yeast Treat the bacterial or yeast cells with lysozyme or lyticase not supplied according the enzyme manufactu
6. have an A260 A280 1 6 and b should be readily digested with restriction enzymes MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com
7. ing or by Proteinase K digestion in AquaGenomic solution About 10 20 ug of genomic DNA can be isolated from 10 mg of tissues IMPORTANT The micelles in AquaGenomic solution may fuse and settle down during storage so vortex vigorously to mix well before dispensing 1 Harvest the Cells Cut out a 2 mm cube 10 mg of frozen or fresh tissue 2 Extract the DNA By homogenization Place the tissue in a pestle and tube homogenizer and homogenize in 100 ul of AquaGenomic solution at room temperature After homogenization add 1 10 volume 10 ul of 100 isopropanol to the sample to reduce forming Briefly vortex and immediately transfer the sample into a 1 5 ml microfuge tube Incubate at room temperature preferable at 60 C for better yield for 4 minutes By Proteinase K digestion Place the tissue in a microfuge tube preloaded with 100 ul of AquaGenomic solution containing 10 ug of Proteinase K Incubate at 60 65 C for 90 minutes and then at 95 C for 10 minutes to inactivate the Proteinase K The tissue is readily disintegrated by vortexing vigorously or pipetting 3 Pellet the debris Vortex the sample vigorously for 30 60 seconds and centrifuge at 10 000 20 000xg for 4 minutes to pellet the debris 4 Pellet the DNA Transfer the clear supernatant 80 ul to a fresh 0 5 ml microfuge tube Add 1 volume of 100 isopropanol and mix the contents by vortexing for 30 seconds Centrifuge at 10 000 20 000xg for 4 minutes to pellet
8. nd 1 volume 100 ul 95 100 of isopropanol Vortex for 30 seconds to mix the contents and centrifuge at 10 000 20 000xg for 4 minutes to pellet the DNA Flip to discard the supernatant as completely as possible Add 200 ul of deionized water pipette and vortex to fully suspend the pellet Centrifuge at 10 000 20 000xg for 4 minutes to pellet the insoluble Transfer the supernatant to a new tube add 3 volumes of 100 ethanol and centrifuge to pellet the DNA Decant to discard the supernatant and fill the tube with 70 ethanol by shooting the ethanol solution at the cap of the tube from a squeeze bottle and then flip to discard the ethanol Repeat the 70 ethanol rinse 2 times Air dry the DNA pellet Add 50 ul of TE buffer or deionized water pipette up and down and vortex vigorously to suspend the DNA Centrifuge again for 2 minutes to pellet any insoluble and transfer the clear DNA solution to a new tube Store the DNA solution at 4 or at 20 C for long term storage MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution Frequently Asked Questions Please read through these questions carefully The answers provide additional helpful tips and useful information for the successful use of AquaGenomic 1 Do I need to store the AquaGenomic kit in the refrigerator or freezer No
9. olution can remove most RNA contaminant Trace amount of RNA would not interfere with most genomic DNA applications However if complete RNA removal is desired RNase A 200 ug ml can be added to the AquaGenomic solution prior to extraction 6 Why do I need to purchase a separate reagent for fecal DNA isolation Due to the presence of large amount of enzyme inhibitors in feces and soil DNA isolation from these samples are particularly challenging Precipitation of the DNA with MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution isopropanol cannot remove some of the inhibitors and a specially formulated AquaPrecipi Solution Product 3015 is required for selective precipitation of the DNA to remove these enzyme inhibitors 7 Can I use water for RBC lysis For fresh blood YES Simply add 10 volumes of deionized water to the blood sample vortex 20 30 seconds centrifuge to pellet the cells remove the red supernatant and then proceed to extract DNA using AquaGenomic For frozen blood NO Thawed RBC will not be lysed with water and you need to use our RBC Lysis Solution Product 4015 which is a modified hypotonic solution that lyses either fresh or frozen RBC 8 Iam concerned about cross contamination using homogenizers any tip Yes Between uses you should thoro
10. res instruction Add 50 ul of 0 5 1 mm glass beads and 100 ul of AquaGenomic Solution to the sample Vortex briefly and incubate the sample at 65 C for 15 minutes to lyse the cells For some strains cell lysis and DNA yield may be enhanced by incubating at 90 C for 30 minutes 3 Pellet the Debris Vortex the sample vigorously for 30 60 seconds and centrifuge at 10 000 20 000xg for 4 minutes to pellet the debris 4 Pellet the DNA Transfer the clear supernatant 90 ul to a fresh 0 5 ml microfuge tube Add 1 volume of 100 isopropanol and mix the contents by vortexing for 30 seconds Centrifuge at 10 000 20 000xg for 4 minutes to pellet the DNA Flip to discard the supernatant Fill the microfuge tube with 70 ethanol by shooting the ethanol solution at the cap of the tube from a squeeze bottle and then flip to discard the ethanol Repeat the 70 ethanol rinse 2 times Flip the tube and blot it several times on a paper towel to remove residual ethanol Air dry the DNA pellet Add 50 ul of TE buffer or deionized water pipette up and down and vortex vigorously to suspend the DNA Centrifuge again for 2 minutes to pellet any insoluble and transfer the clear DNA solution to a new tube Store the DNA solution at 4 or at 20 C for long term storage MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueo
11. rranties of merchantability or fitness for a particular purpose are provided by MultiTarget Pharmaceuticals We shall have no liability for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product Patents Trademarks amp Copyrights AquaGenomic is a trademark of MultiTarget Pharmaceuticals LLC This technology is patent pending 2009 Multitarget Pharmaceuticals LLC All rights reserved MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Cell Protocol This protocol can be used to prepare 5 10 ug of genomic DNA from 1 2 million cultured cells For other preparation scales use 100 ul AquaGenomic Solution for each million nucleated cells IMPORTANT The micelles in AquaGenomic solution may fuse and settle down during storage so vortex vigorously to mix well before dispensing 1 Harvest the Cells Pellet 0 5 2 million cultured cells in a 1 5 ml microfuge tube by centrifugation at 2 000xg for 2 minutes Aspirate or decant to discard the supernatant and leave behind the cell pellet 2 Extract the DNA Add 100 ul of AquaGenomic Solution to the cell pellet Suspend and lyse the cells by vortex vigorously for 30 60 seconds Incubate at room temperature preferable at 60 C
12. the DNA Flip to discard the supernatant Fill the microfuge tube with 70 ethanol by shooting the ethanol solution at the cap of the tube from a squeeze bottle and then flip to discard the ethanol Repeat the 70 ethanol rinse 2 times Flip the tube and blot it several times on a paper towel to remove residual ethanol Air dry the DNA pellet Add 50 ul of TE buffer or deionized water pipette up and down and vortex vigorously to suspend the DNA Centrifuge again for 2 minutes to pellet any insoluble and transfer the clear DNA solution to a new tube Store the DNA solution at 4 or at 20 C for long term storage MultiTarget Pharmaceuticals 2150 West Dauntless Ave Suite 101 Salt Lake City Utah 84116 Phone 1 888 662 6586 Fax 1 801 303 1863 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Tail Protocol Unlike other tissues mouse tail is fibrous therefore Proteinase K not supplied is added to AquaGenomic solution to assist the disintegration of the tissue This protocol uses 100 ul of AquaGenomic solution to prepare 15 20 ug of DNA from 10 mg 2 mm tail snip IMPORTANT The micelles in AquaGenomic solution may fuse and settle down during storage so vortex vigorously to mix well before dispensing 1 Harvest the Tissue Cut off approximately 2 mm long fresh or frozen mouse tail Place the tissue into a microfuge tube preloaded with 100 ul of AquaGenomic solution containing 10 ug of
13. ughly wash the homogenizer with soap and running water soak it in 10 bleach for 5 minutes and then rinse it with running deionized water This will prevent cross contamination of the genomic DNA If you still feel uneasy you can use the 60 C extraction and bead milling method to disrupt the tissues 9 My final DNA pellet does not dissolve well in water what can I do You may soak the final DNA pellet in water or TE buffer at room temperature for 1 16 hours pipette up and down or vortex vigorously to fully disperse the pellet and then centrifuge at 12000xg for 2 minutes to pellet any insoluble 10 What genomic DNA yield can I expect using AquaGenomic Approximately 10 ug DNA can be obtained from 1 2 million cultured cells 300 400 ul of whole blood 1 ml of overnight microbial culture 5 10 mg of animal tissues or 10 20 mg of plant tissues DNA yield is dependent on the number of nucleated cells in the sample and may vary at different cell cycles and for different cell types 11 How pure is the genomic DNA isolated by AquaGenomic Typical A260 A280 of AquaGenomic purified DNA is 1 6 1 8 The isolated genomic DNA is free from most cellular contaminants and enzyme inhibitors However there may be some small RNA contamination if RNase A treatment is not included 12 What QC tests do you use to certify your products Each lot is tested to ensure its performance and reliability in isolating genomic DNA The isolated DNA a should
14. us genomic DNA isolation solution AquaGenomic Stool and Soil Protocol This protocol uses 150 ul of AquaGenomic Solution to prepare 5 10 ug of DNA from 15 mg of feces Isolation of pure DNA from feces or soil is difficult due to the presence of large amount of enzyme inhibitors in these samples The protocol uses an AquaPrecipi Solution Product 3015 not included to remove these PCR inhibitors IMPORTANT The micelles in AquaGenomic solution may fuse and settle down during storage so vortex vigorously to mix well before dispensing 1 Harvest the Cells Weigh out 15 mg of wet feces 10 mg of dry fecal pellet or 30 mg of soil in a 1 5 ml microfuge tube 2 Extract the DNA Add 150 ul of AquaGenomic Solution If mitochondrial DNA extraction is desired add Proteinase K to AquaGenomic to 100 ug ml Incubate at 60 65 C for 90 minutes to digest the mitochondria and then at 95 C for 10 minutes to inactivate the Proteinase K to the sample For dry fecal sample let it soak in AquaGenomic Solution until it is rehydrated Homogenize the sample with a microfuge pestle or bead beater or just vortex vigorously for 1 2 minutes Incubate the sample at 60 80 C for 15 30 minutes 3 Pellet the Debris Vortex vigorously for 30 60 seconds and centrifuge at 10 000 20 000xg for 4 minutes to pellet the debris 4 Pellet the DNA Transfer the supernatant 100 ul to a fresh 1 5 ml microfuge tube Add 0 5 volume 50 ul of AquaPrecipi Solution a
15. ution AquaGenomic Plant Protocol This protocol may be used to isolate 10 20 ug of genomic DNA from 20 mg of plant tissues using 200 ul AquaGenomic Solution IMPORTANT The micelles in AquaGenomic solution may fuse and settle down during storage so vortex vigorously to mix well before dispensing 1 Harvest the Cells Weigh out 20 mg of fresh or frozen plant tissue Cut the tissue into small pieces Place them in 200 ul of AquaGenomic Solution in a pestle and tube homogenizer 2 Extract the DNA Homogenize the tissue at room temperature After homogenization add 1 10 volume 30 ul of 100 isopropanol to the sample to reduce forming Briefly vortex and immediately pour the sample into a 1 5 ml microfuge tube 3 Pellet the debris Centrifuge at 10 000 20 000xg for 4 minutes to pellet the debris 4 Pellet the DNA Transfer the clear supernatant 100 ul to a fresh 1 5 ml microfuge tube Add 1 volume of 100 isopropanol and mix the contents by vortexing for 30 seconds Centrifuge at 10 000 20 000xg for 4 minutes to pellet the DNA Flip to discard the supernatant Fill the microfuge tube with 70 ethanol by shooting the ethanol solution at the cap of the tube from a squeeze bottle and then flip to discard the ethanol Repeat the 70 ethanol rinse 2 times Flip the tube and blot it several times on a paper towel to remove residual ethanol Air dry the DNA pellet Add 50 ul of TE buffer or deionized water pipette up and down and vort
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