AquaGenomic User Manual - MultiTarget Pharmaceuticals
Contents
1. DNA and remove PCR inhibitors However for mouse fecal DNA extraction and genotyping AquaPrecipi purification may not be necessary 1 Harvest the Cells Weigh out 15 mg of wet feces 10 mg of dry fecal pellet or a mouse fecal pellet or 30 mg of soil in a 1 5 ml microfuge tube 2 Extract the DNA Add 200 ul of AquaGenomic solution 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 vortex vigorously for 1 2 min Alternatively homogenize the samples in AquaGenomic in 0 5 ml screw capped tubes with a multichannel bead beater Incubate the sample at 75 C for 20 min If mitochondrial DNA extraction is desired add Proteinase K to AquaGenomic to 100 ug ml Incubate at 55 C for 60 min to digest the mitochondria and then at 95 C for 15 min to inactivate the Proteinase K 3 Pellet the Debris Vortex vigorously for 60 sec and centrifuge at 12 000 xg for 5 min to pellet the debris Transfer the clear lysate 100 ul to a new 0 5 ml microfuge tube Note The lysates of most fecal and soil samples cannot be used directly in PCR reactions as they contain large amounts of PCR inhibitors AquaPrecipi is required for the removal of these fecal and soil PCR inhibitors in the next step 4 Pellet the DNA Add 0 5 vol 50 ul of AquaPrecipi 3015 order separately and 0 5 vol 50 ul of 95 100 of ethanol Vortex for 60 sec and centrifuge at 12 0002. xg for 5 min to pellet the DNA Decant to discard the supernatant Fill the tube with 70 ethanol from a squirt bottle and then flip the tube to discard the ethanol solution Repeat the 70 ethanol rinse once Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 100 ul of TE buffer or deionized water to the DNA pellet pipette or vortex vigorously to suspend the DNA Centrifuge at 12 000 xg for 10 min to pellet any insoluble material which contains residual PCR inhibitors and transfer the clear DNA solution to a new tube MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Swab Protocol Cotton swabs are commonly used in forensic evidence collection However existing DNA extraction methods can recover only 100 500 ng of DNA from a dried specimen swab as the majority of DNA remains entrapped within the cotton matrix With AquaGenomic 5 8 ug of DNA can be extracted from a dried specimen swab It is possible to use cotton swabs for specimen collection transportation storage and DNA extraction for low complexity and low cost biobanking biosurveillance and epidemiological research applications 1 Collect the specimen Use a cotton swab to soak up the specimen 100 200 ul such as blood saliva mucus semen feces cultured mammalian or bacteria cells
3. 9 10 volumes and stored at room temperature until DNA extraction 2 Extract the DNA Add 200 ul of AquaGenomic to 100 ul of lysed avian blood sample in Queen s lysis buffer Vortex vigorously for 60 sec Optional Incubate at 75 C for 20 min for best DNA yield DNase inactivation and RNA degradation 3 Pellet the Debris Add 100 ul of isopropanol diluted AquaRemove order separately 1208 Note Dilute the AquaRemove solution with an equal volume of isopropanol before use and vortex vigorously for 60 sec to mix well Centrifuge at 12 000 xg for 5 min to pellet the debris 4 Pellet the DNA Transfer the clear lysate 400 ul to a new 1 5 ml microfuge tube Add 0 8 vol 320 ul of 100 isopropanol and vortex for 60 sec to mix well Centrifuge at 12 000 xg for 5 min to pellet the DNA Decant to discard the supernatant Fill the tube with 70 ethanol from a squirt bottle and then flip the tube to discard the ethanol solution Repeat the 70 ethanol rinse once Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 100 ul of TE buffer or deionized water to the DNA pellet pipette or vortex vigorously to suspend the DNA MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution Frequently Asked Questions Please read through these questions carefully The answers pr
4. AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Instruction Manual General Information Description AquaGenomic is an aqueous reagent for DNA extraction It may be used to extract DNA from all types of specimens from bacteria to animal tissues The extraction protocols are simple fast and scalable AquaGenomic is nontoxic and its lysate may be used for PCR without further DNA purification AquaGenomic is highly efficient in extracting DNA from dried specimen swabs It enables the use of cotton swabs for specimen collection transportation and storage at room temperature therefore making dried specimen swabs ideal for low complexity and low cost biobanking biosurveillance and epidemiological research applications 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 User Manual 2030 30 ml AquaGenomic Solution User Manual MSDS Available at www aquaplasmid com Storage Store tightly capped at RT 22 C 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 warr
5. I have to use the lysate immediately for PCR No you may store the lysate at 4 C until analysis If the lysate has been incubated at 85 C for 20 min it may even be left at room temperature until analysis 6 I got a weak PCR amplification using the lysate directly how may I improve it You may try a few things to optimize the amplification a try use different amount of lysate for the PCR form 0 25 ul undiluted lysate to 20x diluted lysate b add 0 1 mg ml BSA to the PCR reaction c add 1 mg ml DTT to the PCR reaction and d increase the PCR cycle number to 45 cycles MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com
6. anties of any kind express or implied including without limitation implied warranties 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 2015 Multitarget Pharmaceuticals LLC All rights reserved MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 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 sample sizes use 100 ul of AquaGenomic Solution for each million nucleated cells 1 Harvest the Cells Pellet 0 5 2 million cultured cells in a 1 5 ml microfuge tube by centrifugation at 12 000 xg for 60 sec Aspirate or decant to discard the supernatant 2 Extract the DNA Add 100 ul of AquaGenomic to the cell pellet Suspend and lyse the cells by vortex vigorously for 60 sec Optional Incubate at 75 C for 20 min for best DNA yield DNase inactivation and RNA degradation 3 Pellet the Debris Centrifuge at 12 000 xg for 5 min to pellet the debris Note To use the lysate for PCR dilute an aliq
7. f deionized water and use 0 25 ul of the diluted lysate in a 25 ul PCR reaction 4 Purify the DNA Centrifuge at 10 000 xg for 5 min to pellet any debris in the crude lysate Transfer the clear lysate 200 ul to a 0 5 ml tube and mix with 1 vol of isopropanol 200 ul Centrifuge at 10 000 xg for 5 min to pellet the DNA Flip the tube to discard the isopropanol supernatant as completely as possible Gently shoot 50 isopropanol from a squirt bottle to fill up the tube Flip the tube to discard the isopropanol rinse as completely as possible Tap and place the tube upside down on a clean paper towel to remove residual solution Air dry the DNA pellet for 5 10 min Add 100 ul of deionized water to the DNA pellet and let it rehydrate for gt 15 min Vortex or pipet up and down to solubilize the DNA MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Avian Blood Protocol This protocol may be used to prepare about 50 100 ug of genomic DNA from 10 ul of avian blood sample stored in 100 ul of Queen s lysis buffer 10 mM Tris 10 mM NaCl 10 mM EDTA 1 n lauroylsarcosine pH 7 5 using 200 ul of AquaGenomic solution and 100 ul of AquaRemove solution order separately 1208 1 Collect the blood sample Avian blood samples are routinely collected in Queen s lysis buffer at a ratio of 1 volume to
8. hanol from a squirt bottle and then flip the tube to discard the ethanol solution Repeat the 70 ethanol rinse once Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 100 ul of TE buffer or deionized water to the DNA pellet pipette or vortex vigorously to suspend the DNA Centrifuge at 12 000 xg for 5 min to pellet any insoluble material and transfer the clear DNA solution to a new tube MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Whole Blood Protocol AquaGenomic may be used to extract total blood cellular and cell free DNA from whole blood samples and its lysate may be used directly for PCR without further purification a To use lysate directly for PCR 1 Add 20 ul of AquaGenomic solution to each well in a 0 2 ml 96 well PCR plate 2 Transfer 10 ul of well mixed fresh or thawed whole blood to the AquaGenomic solution Pipet up and down a few times to mix 3 Incubate at 75 C for 20 min in a PCR machine with heated lid After the incubation add 170 ul of water to the blood clot Shake or pipet up and down a few times to mix 4 To amplify the DNA add 0 25 ul of the diluted lysate to 25 ul of PCR master mix and subject the reaction mix to 35 45 cycles of PCR amplification b To purify total blood DNA 1 Add 200 ul of AquaGenomic so
9. homogenized animal or plant tissues or any other potential sources of biospecimens Air dry the specimen swab at ambient temperature 20 50 C for gt 24 hours The dried specimen swabs can be shipped at ambient temperature and then stored at room temperature in sealed paper envelopes or plastic bags with desiccant for many years 2 Extract the proteins and small molecules dried blood swabs Cut off the specimen swab tip into a 1 5 ml microfuge tube Add 400 ul deionized water or buffer and soak the swab for gt 30 min Use a 1 ml pipet tip to smash the swab 10 times and press it to the bottom of the tube to squeeze out the solution Transfer as much liquid as possible 300 ul to a new 1 5 ml microfuge tube for analysis of plasma proteins or small molecules 3 Extract the DNA Add 300 ul AquaGenomic to the 1 5 ml tube containing the wet swab from Step 2 or a new dried specimen swab Incubate at 22 C for 1 hr and then at 75 85 C for 20 min Use a 1 ml blue pipet tip to smash the swab 10 times and press it to the bottom of the tube to squeeze out the solution Alternatively homogenize the samples in AquaGenomic in 0 5 ml screw capped tubes with a multichannel bead beater Transfer as much liquid as possible 200 ul to a new 0 5 ml microfuge tube or use a microfuge spin bucket to recover all the lysate from the swab to maximize the DNA yield Optional To use the crude lysate for PCR dilute an aliquot of the crude lysate with 10 20 vol o
10. ization Homogenize the tissue in 200 ul of AquaGenomic Move the pestle up and down slowly while vortexing at top speed to enhance homogenization Alternatively homogenize the tissue in 0 5 ml screw capped tubes with a multichannel bead beater After homogenization add 1 10 volume 10 ul of isopropanol to the sample to reduce foaming do not add isopropanol prior to homogenization as it will neutralize AquaGenomic s cell lysis ability vortex and transfer the homogenate to a 1 5 ml microfuge tube Optional Incubate at 75 C for 20 min for best DNA yield DNase inactivation and RNA degradation By Proteinase K digestion Place the tissue e g a mouse tail clip in a microfuge tube preloaded with 200 ul of AquaGenomic containing 10 ug of Proteinase K Incubate at 55 C for gt 90 min to digest the tissue and then at 95 C for 10 min to inactivate the Proteinase K The tissue is readily disintegrated by vortexing or pipetting 3 Pellet the debris Centrifuge at 12 000 xg for 5 min to pellet the debris Note To use the lysate for PCR dilute an aliquot of the lysate with 10 20 vol of water and then use 0 25 ul of the diluted lysate in a 25 ul PCR reaction 4 Pellet the DNA Transfer the clear lysate 180 ul to a new 0 5 ml microfuge tube Add 0 8 vol 144 ul of 100 isopropanol and vortex for 60 sec to mix well Centrifuge at 12 000 xg for 5 min to pellet the DNA Decant to discard the supernatant Fill the tube with 70 et
11. lution to a 0 5 ml tube 2 Transfer 100 ul of well mixed fresh or thawed whole blood to the AquaGenomic solution Pipet up and down a few times to mix 3 Incubate at 75 C for 20 min After the incubation centrifuge the samples at 12 000 xg for 5 min Transfer the clear lysate 200 ul to a new 0 5 ml microfuge tube 4 Add 0 8 vol 160 ul of 100 isopropanol to the clear lysate and vortex for 60 sec to mix the contents Centrifuge at 12 000 xg for 5 10 min at 22 C to pellet the DNA Decant to discard the supernatant Carefully fill the tube with 70 ethanol from a squirt bottle and then flip the tube to discard the ethanol solution Repeat the ethanol rinse once 5 Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 50 ul of TE buffer or deionized water to the DNA pellet vortex vigorously to suspend the DNA MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Saliva Protocol Approximately 10 20 ug of genomic DNA can be obtained from 50 ul of saliva or a buccal swab or 200 ul of mouthwash using one of the following methods 1 Harvest the Cells a Saliva Swirl and rub your tongue against the inside of your cheek and gum for 5 10 times Carefully spit the saliva into a clean weight boat or a 15 ml conical tube b Swab Use a swab to
12. n Repeat the 70 ethanol rinse once Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 100 ul of TE buffer or deionized water to the DNA pellet pipette or vortex vigorously to suspend the DNA MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Microbe Protocol This protocol can be used to prepare 10 20 ug of DNA from 1 ml overnight microbial culture For other preparation scales use 100 ul of AquaGenomic Solution for each milliliter of overnight culture 1 Harvest the Cells Centrifuge 1 ml overnight bacterial culture at 12 000xg for 60 sec to pellet the cells Aspirate or decant to discard the supernatant 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 sec Optional Incubate at 75 C for 20 min for best DNA yield DNase inactivation and RNA degradation Alternatively homogenize the samples in AquaGenomic in 0 5 ml screw capped tubes with a multichannel bead beater For Gram positive bacteria or yeast Treat the bacterial or yeast cells with lysozyme or lyticase not supplied according the enzyme manufactures instruction Add 50 ug of 0 5 1 mm glass beads and 100 ul of AquaGenomic Solution containing 100 ug ml Proteinase K to the
13. ovide additional helpful tips and useful information for the successful use of AquaGenomic 1 Do I need to keep AquaGenomic in the freezer No AquaGenomic Solution is stable at room temperature 22 C for gt 1 year 2 Does AquaGenomic Solution contain Proteinase K No AquaGenomic can be used to extract DNA from most cells and tissues without needing protease digestion However adding Proteinase K 50 ug ml to AquaGenomic solution can increase DNA yield and is required for mitochondrial DNA extraction You may homogenize the sample in AquaGenomic containing Proteinase K incubate it at 55 C for 1 2 hrs and then at 95 C for 10 15 min to inactivate the Proteinase K 3 AquaGenomic sounds like a green product any particular precaution AquaGenomic is nontoxic and non corrosive It contains no phenol chloroform guanidine HCl or other harmful chemicals commonly used for DNA extraction There is no particular precaution while using AquaGenomic you just need to follow standard good laboratory practice in handling laboratory chemicals 4 I am worried about cross contamination using homogenizers any tips Between uses you may wash the homogenizer with soap and running water soak it in 10 bleach for 5 min and then rinse it with running deionized water If you still feel uneasy you may use Proteinase K digestion without using a homogenizer or use a multichannel bead beater for homogenization in screw capped tubes 5 Do
14. rub the inside of your cheek and gum for 5 10 times and let it soak up the saliva Air dry the swab in its pouch c Mouthwash Swirl and rub your tongue against the inside of your cheek and gum for 5 10 times Rinse the mouth with 10 20 ml of Scope mouthwash and spit it into a 50 ml conical tube 2 Extract the DNA a Saliva Transfer 50 ul of saliva to a microfuge tube preloaded with 100 ul of AquaGenomic solution and incubate at 75 C for 20 min b Swab Cut off the tip of the swab into a 1 5 ml microfuge tube Add 300 ul of AquaGenomic solution and incubate at 75 C for 20 min Use a 1 ml pipet tip to smash the swab 10 times to the bottom of the tube to squeeze out of the liquid c Mouthwash Centrifuge 200 ul of mouthwash at 10 000 xg for 5 min to pellet the buccal cells and discard the supernatant Add 200 ul of AquaGenomic solution Vortex to mix well 3 Pellet the Debris Centrifuge at 12 000 xg for 5 min to pellet the debris Note To use the lysate for PCR dilute an aliquot of the lysate with 10 20 vol of water and then use 0 25 ul of the diluted lysate in a 25 ul PCR reaction 4 Pellet the DNA Transfer the clear lysate 100 ul to a new 0 5 ml microfuge tube Add 0 8 vol 80 ul of 100 isopropanol and vortex to mix well Centrifuge at 12 000 xg for 5 min to pellet the DNA Decant to discard the supernatant Fill the tube with 70 ethanol from a squirt bottle and then flip the tube to discard the ethanol solutio
15. sample Incubate at 55 C for 60 min and then at 95 C for 10 min to inactivate the Proteinase K 3 Pellet the Debris Centrifuge at 12 000 xg for 5 min to pellet the debris Note To use the lysate for PCR dilute an aliquot of the lysate with 10 20 vol of water and then use 0 25 ul of the diluted lysate in a 25 ul PCR reaction 4 Pellet the DNA Transfer the clear lysate 90 ul to a new 0 5 ml microfuge tube Add 0 8 vol 72 ul of 100 isopropanol and vortex for 60 sec to mix well Centrifuge at 12 000 xg for 5 min to pellet the DNA Decant to discard the supernatant Fill the tube with 70 ethanol from a squirt bottle and then flip the tube to discard the ethanol solution Repeat the 70 ethanol rinse once Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 100 ul of TE buffer or deionized water to the DNA pellet pipette or vortex vigorously to suspend the DNA Centrifuge at 12 000 xg for 5 min to pellet any insoluble material and transfer the clear DNA solution to a new tube MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Stool and Soil Protocol This protocol uses 200 ul of AquaGenomic Solution to prepare 5 10 ug of DNA from 15 mg of feces AquaPrecipi solution Item 3015 not included is generally required to purify fecal and soil
16. uot of the lysate with 10 20 vol of water and then use 0 25 ul of the diluted lysate in a 25 ul PCR reaction 4 Pellet the DNA Transfer the clear lysate 90 ul to a new 0 5 ml microfuge tube Add 0 8 vol 72 ul of 100 isopropanol and vortex for 60 sec to mix well Centrifuge at 12 000 xg for 5 min to pellet the DNA Decant to discard the supernatant Fill the tube with 70 ethanol from a squirt bottle and then flip the tube to discard the ethanol solution Repeat the 70 ethanol rinse once Place the tube upside down on a clean paper towel for 5 10 min to air dry the DNA pellet Add 100 ul of TE buffer or deionized water to the DNA pellet pipette or vortex vigorously to suspend the DNA Centrifuge at 12 000 xg for 5 min to pellet any insoluble material and transfer the clear DNA solution to a new tube MultiTarget Pharmaceuticals 5050 Edison Ave Ste 214 Colorado Springs CO 80915 USA 801 769 6586 www aquaplasmid com AquaGenomic an aqueous genomic DNA isolation solution AquaGenomic Tissue Protocol This protocol may be used to extract DNA from tissues including common research specimens such as drosophila mouse tail snip nematode zebrafish and plant tissues by homogenization or by Proteinase K digestion in AquaGenomic solution Approximately 10 20 ug of DNA can be extracted from 10 mg of animal tissue 1 Harvest the Cells Cut out a 2 mm cube 10 mg of frozen or fresh tissue 2 Extract the DNA By homogen
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