Contents - Biomiga
Contents
1. 3 Carefully remove the supernatant leaving 1 mL of pellet and Buffer A 4 Vortex for 5s and centrifuge at full speed to collect any sample adhering to the walls of tubes Transfer the sample to a 2 0 mL tube 5 Add 0 5 mL of Buffer A to the Corex tube and vortex for 30s and centrifuge at full speed to collect any sample adhering to the walls of the tube Transfer sample to a 2 mL microfuge tube 6 Centrifuge for 2 min at full speed 7 Carefully remove the supernatant without disturbing the semen pellet 8 Resuspend pellet in 200 uL of Buffer B 9 Add 25 uL Proteinase K 25 mg mL and incubate for 2 hours at 60 10 Invert the tube occasionally to disperse the sample or place on a rocking platform Page 6 Biomiga EZgene Forensic gDNA Kit 11 Add 250 pL Buffer BL and 260 pL ethanol to the sample and mix by vortexing 12 Go to step 6 on page 4 and follow the standard protocol to carry out the DNA purification Page 7 Biomiga EZgene Forensic gDNA Kit Protocol for Isolation of Genomic DNA from Buccal Swabs Typical yields from cotton or C E P swabs are typically around 0 5 3 ug DNA 1 Follow standard protocol scrape the swabs firmly against the inside of cheek 5 10 times Air dry or vacuum the swabs for 1 2 hours after collection The person providing the sample should not eat or drink for at least 30 min prior to the sample collection Remove and transfer the buccal swab into a 2 0 mL tube and a2. 0 rpm for 1 min to elute DNA Note Add the eluted DNA back to the column for a second elution will yield another 20 of DNA bound Note Incubation at 70 C rather than at room temperature will give a modest increase in DNA yield Protocol for Isolation of Bacterial gDNA from Biological Fluids 1 Pellet bacteria by centrifuging 10 min at 8 000 rpm 2 Resuspend the pellet with 200 uL Buffer TL 3 Follow the standard protocol Page 4 from Step 3 Protocol for Isolation of Genomic DNA from Eye Nasal and Other Swabs Collect the sample and add 2 mL PBS Incubate 2 3 hours at 30 C 2 Pellet bacteria by centrifuging 10 min at 8 000 rpm 3 Resuspend bacterial pellet with 200 uL Buffer TL 4 Follow the standard protocol Page 4 from Step 3 Page 9 Biomiga EZgene Forensic gDNA Kit Protocol for Isolation of DNA from Salvia 6 7 Addl 5 mL saliva to a 15 mL centrifuge tube contains 6 mL PBS Vortex to mix well Centrifuge at 2000 x g for 5 min Discard the supernatant and ressuspend the pellet in 180 uL PBS Transfer the sample to a new 1 5 mL centrifuge tube Add 25 pL Protease K solution 200 uL Buffer BL Add 20 uL RNase A solution if RNA free DNA is desired Mix throughly by vortexing for 10s Briefly centrifuge to collect any droplets from the lid Incubate 15 min for 70 C with occasional mixing Add 200 uL absolute ethanol and mix throughly by voretxing Go to Step 6 on page 4 and ca
3. 00 mL Buffer KB 3 mL 28 mL 135 mL DNA Wash Buffer 2 mL 15 mL 3 x 24 mL Elution Buffer 2 mL 15 mL 60 mL Protease K 3 mg 30 mg 5 x 30 mg User Manual 1 1 1 For isolation gDNA from sperm Product GD2512 00 GD2512 01 GD2512 02 10x Buffer A 5S mL 50 mL 250 mL Buffer B 1 mL 15 mL 70 mL Note The kit is supplied with enough buffers for the standard protocol However due to increased volumes called for in some protocols such as the buccal swab protocol extra buffers can be purchased separately from Biomiga See product inforamtion on our website or call customer service for price information Before Starting Important Reconstitute Protease K in 110 uL GD2512 00 or 1 3 mL GD2512 01 or 5 x 1 3 mL GD2512 02 Elution Buffer Vortex the vial and spin the vial briefly prior to use Dilute DNA Wash Buffer with absolute ethanol as follows e GD2512 00 Add 8 mL ethanol e GD2512 01 Add 60 mL ethanol e GD2512 02 Add 96 mL ethanol bottle e Prepare DL Dithiothreitol DTT for Hair Nails and Feathers protocol Page 3 Biomiga EZgene Forensic gDNA Kit Protocol for Isolation of DNA from Dried Blood Body Fluids and Sperm Spots Dried blood body fluids and sperm samples on filter paper can be processed using the following method Use Specimen paper or blot paper for spotting samples as the filter paper disintegrates DNA when aqueous buffers are added 1 Punch out the sample spot from the filter paper Use le
4. 13 000 rpm to dry the column This step is critical for removing residual ethanol that might interfere with DNA yield and purity 11 Place the column into a sterile1 5 mL tube and add 100 uL of preheated 70 C Elution Buffer Incubate the tube at 70 C for 3 minutes 12 Centrifuge at 10 000 rpm for 1 min to elute the DNA Note Add the eluted DNA back to the column for a second elution will yield another 2096 of DNA bound Note Incubation at 70 C rather than at room temperature will give a modest increase in DNA yield per elution Note Blood spots from finger pricks usually contain no more than 50 uL blood and yield approximately 0 5 1 ug DNA This is sufficient for PCR analysis Page 5 Biomiga EZgene Forensic gDNA Kit Protocol for Isolation of Genomic DNA from Sperm This protocol can be used for fresh or frozen semen samples with equal efficiency Frozen samples must to be thawed thoroughly before use Note that lysis time will vary depending on the size and density of the source material Dilute the following buffer before starting Buffer A Dilute 10x Buffer A to 1x Buffer A with ddH O before use Buffer B Add B mercaptoethanol to final percentage of 2 v v 1 Add 50 250 uL of sperm to 10 mL of Buffer A in a glass Corex centrifuge tube Vortex for 10 seconds at full speed Only use Corex tubes to prevent attachment of the sperm cells to the tube walls 2 Centrifuge for 10 min at 4 000 rpm 2500 x g
5. Contents Introduction enrenar eee mee ees 2 Storage and Stability csse eese ette entente 2 Safety Informations iia o AR Kit Contents eeu tet t iets ne et te etti ceste dd 3 Before Starting ie sce ANA NANANA I bleed DNA NANA 3 Protocol for Dried Body Fluid Samples sess 4 Protocol for DNA Isolation from Sperm s 6 Protocol for Buccal Swabs eee 8 Protocol for Bacterial DNA From Biological Fluids 9 Protocol for Eye Nasal and Other Swabs sess 9 Protocol for Salva 4 AA LANA getestet 10 Protocol for Hair Nails and Feathers cccccccceessseesssssseeeseeeeeees 10 Vacuum Spin Protocol ana eredi etie tite dete tee 11 Determination of Yield and Quality sess 12 Trouble Shooting Guide eese 13 Related Products cos ee ethene Ge sp Saves ERR ERO EE S ERE ENTE 15 Limited Use and Warranty 0cc ccc eceecee cnet neces ene eneene ones 16 Page 1 Biomiga EZgene Forensic gDNA Kit Introduction The EZgene Forensic gDNA Purification Kit provides a rapid and easy method for the isolation of genomic DNA from forensic samples such as dry blood buccal swabs and sperm for consistent PCR and Southern analysis This kit can also be used for the preparation of genomic DNA from mouse tail snips whole blood buffy coat serum and p
6. dd to the tube Add 525 uL PBS 25 pL Protease K solution and 525 uL Buffer BL to the sample Mix immediately by votexing for 30s Incubate 30 min at 60 C with occasional mixing Briefly centrifuge to remove any droplets from inside the lid Add 525 uL absolute ethanol and mix thoroughly by vortexing Briefly centrifuge to collect any droplets from the lid Carefully apply 600 uL of the mixture into the column Centrifuge at 10 000 rpm for 30s Discard the flow through liquid and put the column back to the collection tube Carefully apply the remaining mixture to the column and centrifuge at 10 000 rpm for 30s Discard the flow through liquid and collection tube Put the column into another collection tube Add 650 uL of DNA Wash Buffer and centrifuge at 10 000 rpm for 1 min Discard the flow through liquid and put the column back to the collection tube Add 650 uL of DNA Wash Buffer and centrifuge as above Discard the flow through liquid and collection tube Put the column with the lid open to a new collection tube Centrifuge at 13 000 rpm for 2 min to dry the column This step is critical for removal of residual ethanol that might result in decreased DNA yield and purity Page 8 Biomiga EZgene Forensic gDNA Kit 10 Place the column into a nuclease free 1 5 mL microfuge tube and add 100 pL of preheated 70 C Elution Buffer Allow tube to incubate at 70 C for 3 min 11 Centrifuge at 10 00
7. his product is warranted to perform as described in its labeling and in Biomiga s literature when used in accordance with instructions No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness for a particular purpose are provided by Biomiga Biomiga s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of Biomiga to replace the products Biomiga shall have no liability for any direct indirect consequential or incidental damage arising out of the use the results of use or the inability to use it product For technology support or learn more product information please visit our website at www biomiga com or contact us at 858 603 3219 FOR REACHER USE ONLY Page 16 Biomiga EZgene Forensic gDNA Kit
8. lasma No phenol chloroform extractions and isopropanol or ethanol precipitation are needed DNA purified using this method is ready for downstream applications such as PCR Southern blotting and restriction digestion In this procedure samples are first lysed and applied to the spin column that DNA binds While cellular debris hemoglobin and other proteins are effectively washed away by DNA Wash Buffer pure DNA is eluted in sterile deionized water or elution buffer Each ezBind column can bind approximately 100 ug DNA Storage and Stability Once reconstituted Protease K must be stored at 20 C All other components can be stored at 22 25 C Under these conditions performance of all components of the kit are guaranteed at least 12 months from the date of purchase Under cool ambient conditions a precipitate may form in the Buffer BL heat the bottle at 37 C to dissolve the precipitate before use Safety Information Buffer BL contains acidic acid and chaotropic salts which may form reactive compounds when combines with bleach Do not add bleach or acidic solutions directly to the preparation waste Ware gloves and protective eyeware when handling this solution Page2 Biomiga EZgene Forensic gDNA Kit Kit Contents Product GD2512 00 GD2512 01 GD2512 02 Preps 4 50 250 DNA Columns 4 50 250 2 mL Collection Tubes 8 100 500 Buffer BL 3 mL 30 mL 150 mL Buffer TL 3 mL 20 mL 1
9. m for 1 min to dry the column It is critical to remove the residual ethanol completely for optimal DNA yield and purity Place the column in a nuclease free 1 5 mL microcentrifuge tube and add 50 100 pL Elution Buffer or ddH O Allow standing for 1 2 min and then centrifuge 1 min to elute DNA Note Add the eluted DNA back to the column for a second elution will yield another 20 of DNA bound Note Incubation at 70 C rather than at room temperature will give a modest increase in DNA yield Page 11 Biomiga EZgene Forensic gDNA Kit Determination of Yield and Quality The total DNA yield can be determined by DNA Absorbance 9 x 0 05 ug uL x Dilution factor The quality of DNA can be evaluated by OD 260 280 A ratio of Aoso A so of 1 8 1 9 corresponds to 9096 9596 purity Expected yields vary with both amount and type of tissue used 30 mg of fresh tissue will yield approximately10 40 ug DNA with two elutions each 100 uL Page 12 Biomiga EZgene Forensic gDNA Kit Trouble Shooting Guide Problem Possible Cause Suggestions Forgot to add Before applying sample to column both Buffer BL ethanol and ethanol must be added See protocol above Forgot to add Dilute DNA Wash Buffer with the indicated ethanol to DAN volume of absolute ethanol before use Colored Wash Buffer residue in Incomplete lysis Buffer BL is viscous and the sample must be column due t
10. o improper vortexed thoroughly After mixing with Buffer washing BL No ethanol added Dilute DNA Wash Buffer with the indicated to DNA Wash volume of absolute ethanol before use Buffer Concentrate Incomplete lysis Extend incubation time of lysis with Buffer TL and protease Add the correct volume of Buffer BL and incubate for specified time at 70 C It may be necessary to extend incubation time by 10 min Column Sample too large If using more than 30 mg tissue increase volumes clogged of Proteinase K Buffer TL Buffer BL and ethanol Pass aliquots of lysate through one column successively Sample too viscous Divide sample into multiple tubes adjust volume to 250 uL with 10 mM Tris HCl Clogged column See above Poor sample Incubate the OB specimen collection paper longer release from in TL buffer Shake the tubes frequently collection paper Low DNA Poor elution Repeat elution or increase elution volume yield Incubation of column at 70 C for 5 min with Elution Buffer may increase yields Improper washing DNA Wash Buffer Concentrate must be diluted with absolute 100 ethanol as specified on Page 3 before use Page 13 Biomiga EZgene Forensic gDNA Kit Trouble Shooting Guide Continue from page 13 Problems Possible Cause Suggestions Extended Resin from the column may be present in eluate centrifugation Avoid centrifugation at speeds higher than during elution s
11. rry out the purification procedure Protocol for Isolation of DNA from Hair Nails and Feathers 1 Cut the sample into small pieces 0 5 1 cm and transfer to a 1 5 mL centrifuge tube Note For hair cut from base of hair for feathers select the primary feathers Large birds secondary tail or breast feather can be used 2 Add 250 uL TL Buffer 25 uL Protease K and 20 pL 1 M DTT Mix thoroughly by vortexing Incubate 30 min at 60 C with occasional mixing 3 Add 250 uL Buffer BL to the sample mix throughly by vortexing Add 250 uL absolute ethanol to the sample mix thoroughly by vortexing 4 Follow the standard protocol from Step 6 on Page 4 Page 10 Biomiga EZgene Forensic gDNA Kit Vacuum Spin Protocol Go through the previous sections of this manual before using this protocol 1 Prepare samples by following the standard protocol in previous sections Steps 1 5 Prepare the vacuum manifold according to manufacturer s instructions and connect the column to the manifold Load the sample Buffer BL ethanol mixture into the column Switch on vacuum source to draw the sample through the column then turn off the vacuum Wash the column by adding 650 uL DNA Wash Buffer Draw the DNA Wash Buffer through the column by turning on the vacuum source Repeat this step with another 650 uL DNA Wash Buffer Transfer the column with the lid open to a 2 mL collection tube Centrifuge at 13 000 rp
12. ss than 200 uL of blood each spot Tear or cut filter into small pieces and place into a microfuge tube Note Use 3 mm diameter cycle puncher for each sample 2 Add 200 pL Buffer TL and incubate at 55 C for 15 20 min Mix well by vortexing 3 5 times during incubation 3 Add 25 pL Protease K solution and mix well by votexing Incubate for at 60 C for 30 60 min with occasional mixing Spin the sample briefly to collect droplets from the cap 4 Add 225 pL Buffer BL and mix well by vortexing Incubate at 60 C for 10 min Spin the sample briefly to collect an droplets from the cap 5 Add 225 uL absolute ethanol and mix thoroughly by vortexing Briefly centrifuge to remove any droplets inside the tube 6 Apply the entire sample into a DNA column including any precipitate that may have formed Centrifuge at 10 000 rpm for 1 min Discard collection tube and flow through liquid 7 Transfer the column into a new collection tube and add 500 uL of Buffer KB into column Centrifuge at 10 000 rpm for 30s Dispose of flow through liquid and re use the collection tube 8 Add 650 uL of DNA Wash Buffer Centrifuge at 10 000 rpm for 30s Discard the flow through liquid and put the column back to the collection tube 9 Add 650 uL of DNA Wash Buffer and centrifuge as above Discard flow through and put the column with the lid open to a new collection tube Page 4 Biomiga EZgene Forensic gDNA Kit 10 Centrifuge at the column at
13. tep specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digests Poor cell lysis due to incomplete mixing with Buffer Repeat the procedure this time making sere to vortex the sample with Buffer BL immediately and completely A20 A230 BL ratio lower than 13 Incomplete cell Increase incubation time with Buffer TL and lysis or protein protease Ensure that no visible pieces of tissue degradation due to remain insufficient incubation Samples are rich in After applying to column wash with 300 uL ofa protein 1 1 mixture of Buffer BL and ethanol and then with DNA Wash Buffer Poor cell lysis due Mix thoroughly with Buffer BL prior to loading No DNA jos etch to improper mixing ezBind column with Buffer BL Page 14 Biomiga EZgene Forensic gDNA Kit Related EZgene Products Catalog Product Name Preps Price GD2211 02 25 420 00 GD2311 02 25 420 00 GD2411 02 2 495 00 GD2412 02 2 420 00 GD2413 02 2 420 00 GD2414 02 2 420 00 GD2415 02 2 420 00 GD2416 02 25 420 00 eoe Alolalo 4 o nn Qi Qi Qi uA Aika 2 gt Reo 5 Aka Zo GD2512 01 Forensic gDNA purification kit 85 00 GD2512 02 Forensic gDNA purification kit 250 380 00 Page 15 Biomiga EZgene Forensic gDNA Kit Limited Use and Warranty This product is intended for in vitro research use only Not for use in human T
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