How can we extract gDNA(genomic DNA)?
what is the procedure of extraction?what are the appllications of gDNA?
- Detergent and salt in water to extract. Precipitate with ethanol. Dry and resuspend in H2O. Done.00
- If you're looking to find a specific part, you could use PCR. If you want to see the specific make-up of the DNA, then you should use gel electrophoresis00
- The following steps are employed to isolate DNA from eukaryotic cells
1- Disruption of cell membranes by homogenization .
2- Isolation of nuclei by centrifugation .
3- Disruption of nuclear membranes and denaturation of nucleoproteins by action of deter agents.
4-Precipitation and removal of denaturated proteins.
5-Precipitation and removal of DNA .
After that u can make PCR to coplementary strands of DNA .00
- Hahahahahaha. As soon as I stop laughing at the section [sculpture, what the ****?] I will answer... Well, I am an avid CSI fan [well, I was... and then Grissom left. Now it sucks. Lawrence Fishburn, seriously?]. And, I can tell you from my superior knowledge of Crime Scenes, you cannot get DNA from poop. I am sorry. But, you already got a confession from Princess... so, it's all good. [I wish I could star this].00
- Genomic DNA Extraction Methods
Preparation of Genomic DNA from Mammalian Tissue
Purification of DNA using Glass Beads
Phenol Extraction and Ethanol Precipitation of DNA
Preparation of Genomic DNA from Mammalian Tissue
Excise an immediately mince tissue quickly and freeze in liquid nitrogen.
Grind 200 mg to 1g tissues with prechilled mortar and pestle, or crush with hammer to fine powder. Suspend in 1.2 ml digestion buffer per 100mg of tissue.
Tissue Culture Cells
Centrifuge cells 5 minutes at 500xg and discard supernatant. Trypsinize adherent cells first.
Resuspend cells in 1 to 10 ml ice-cold PBS. Centrifuge 5 minutes at 500xg, discard supernatant, and repeat.
Resuspend cells in 1 volume digestion buffer.
Incubate samples, shaking, in tightly capped tubes, 12 to 18 hours at 50C
Extract samples with an equal volume of phenol/chloroform/isoamyl alcohol. Centrifuge 10 minutes at 1700xg. If phases do not resolve well, add another volume digestion buffer, ommiting proteinase K, and repeat centrifugation. If thick white material appears at interface, repeat organic extraction. Transfer top layer to a new tube.
Add ½ volume of 7.5M ammonium acetate and 2 volumes of 100% ethanol. Centrifuge 2 minutes at 1700 xg.
(to prevent shearing of high molecular weight DNA, remove organic solvents and salt by two dialyses against 100 volume TE buffer for >24 hours; omit step 6)
Wash with 70% ethanol, air dry, and resuspend in TE buffer at ~1mg/ml.
(remove residual RNA by adding 0.1% SDS and 1ug/ml Dnase-free Rnase, incubating 1 hour at 37C, and repeating steps 4 and 5)
Purification of DNA using Glass Beads
Add 3 vol 6M NaI solution to DNA in a 1.5 ml microcentrifuge tube. Add glass beads suspension as follows: for amounts of DNA <5ug, use 5 ul glass beads suspension; for amounts of DNA >5ug, use 5ul plus an additional 1 ul for each 0.5 ug increment above 5 ug. Incubate 5 minutes at room temperature.
Microcentrifuge DNA/glass beads complex 5 seconds. Remove and discard supernatant.
Wash the DNA/glass beads pellet three times with 500ul wash solution. Lightly vortex the mixture to resuspend the beads, then microcentrifuge briefly to pellet the beads.
Resuspend pellet in TE buffer, pH 8.0, at 0.5 ug/ul. Incubate 2 to 3 minutes at 45C to elute DNA from the glass beads.
Microcentrifuge 1 minute and transfer the DNA-containing supernatant to a clean tube. Store at 4C until use.
Phenol Extraction and Ethanol Precipitation of DNA
Add an equal volume of phenol/chloroform/isoamyl alcohol to the DNA solution to be purified in 1.5 ml microcentrifuge tube.
Vortex vigorously 10 seconds and microcentrifuge 15 seconds at room temperature.
Carefully remove the top aqueous phase containing the DNA using a 200 ul pipettor and transfer to a new tube. If a white precipitate is present at the aqueous/organic interface, reextract the organic phase and pool aqueous phases.
Add 1/10 volume of 3M sodium acetate, pH 5.2, to the solution of DNA. Mix by vortexing briefly or by flicking the tube several times with your finger.
Add 2 to 2.5 volumes of ice cold 100% ethanol. Mix by vortexing and place in crushed dry ice for 5 minutes or longer.
Spin 5 minutes in a microcentrifuge at high speed and remove the supernatant.
Add 1 ml of room temperature 70% ethanol. Invert the tube several times and microcentrifuge as in step 6.
Remove the supernatant. Dry the pellet in a desiccator under vacuum or in a hood.
Dissolve the dry pellet in an appropriate volume of water or TE buffer, pH8.0
Preparation of Plant DNA using CTAB
Add 2-mercaptoethanol to the required amount of CTAB extraction solution to give a final concentration of 2% (v/v). Heat this solution and CTAB/NaCl solution to 65C.
(4 ml of 2-mercaptoethanol/CTAB solution and 0.4-0.5 ml CTAB/NaCl solution for each gram of leaf tissue)
Chill a pulverizer/homogenizer with liquid nitrogen or dry ice. Pulverize plant tissue to a fine powder and transfer the frozen tissue to an organic solvent-resistant test tube or beaker.
Add warm 2-ME/CTAB-extraction solution to the pulverized tissue and mix to wet thoroughly. Incubate 10 to 60 minutes at 65C with occasional mixing.
Extract the homogenate with an equal volume of 24:1 chloroform/octanol or chloroform/isoamyl alcohol. Mix well by inversion. Centrifuge 5 minutes at 7500 xg, 4C. Recover the top phase.
Add 1/10 volume 65C CTAB/NaCl solution to the recovered aqueous phase and mix well by inversion.
Extract with an equal volume of chloroform/octanol. Mix, centrifuge, and recover top phase.
Add exactly 1 volume CTAB precipitation solution. Mix well by inversion. If precipitate is visible, proceed to step 8. If not, incubate mixture 30minutes at 65C.
Centrifuge 5 minutes at 500xg, 4C.
Remove but do not discard the supernatant and resuspend pellet in high-salt TE buffer (0.5 to 1 ml per gram of starting material). If the pellet is difficult to resuspend, incubate 30 minutes at 65C. Repeat until all or most of pellet is dissolved.
Precipitate the nucleic acids by adding 0.6 volume isopropanol. Mix well and centrifuge 15 minutes at 7500xg, 4C.
Wash the pellet with 80% ethanol, dry, and resuspend in a minimal volume of TE buffer (0.1 to 0.5 ml per gram of starting material).
Whatman FTA is an excellentDNA storage and preparationmedium for samples to beamplified by strand displacement amplification.Whatman FTA®products are powerfultools that simplify the collection, shipment, storage and purification ofnucleic acids from a wide variety ofsources. Animal, plant or microbe cellularsamples are applied directly onto an FTACard, activating chemicals in the card thatlyse the cells, inactivate proteins andimmobilize the genomic nucleic acids.Infectious organisms are inactivated in theprocess. After drying, the nucleic acids areprotected from enzymatic, microbial,oxidative and free-radical degradation.The samples can be stored for years atroom temperature before analysis (14 years to date for blood on FTA). For analysis or use of the stored DNA asmall disk is cut from the sample area onFTA. Washing with FTA PurificationReagent removes contaminants. DNAimmobilized in the washed disk can thenbe amplified by polymerase chain reaction(PCR), cut by restriction endonucleases orused in other ways. For some applications,however, larger amounts of DNA areneeded than the disk—or even the wholeoriginal sample—contains. They may alsorequire more sections of the sequencethan even multiplex PCR can supply.These applications have in the pastrequired isolation of DNA from largersamples. Whole genome amplification(WGA) has been developed to supplyessentially unlimited quantities of DNAfrom small samples. This application notedescribes the use of strand displacementamplification, a popular WGA method, to generate micrograms of DNA from asmall sample of human blood cell DNApreserved on FTA.The GenomiPhi™ DNA Amplification Kitfrom Amersham Biosciences uses Phi29DNA polymerase and a multiple stranddisplacement mechanism to amplify smallinitial amounts of linear template DNA. It produces microgram quantities of highmolecular weight copies that representthe whole template sequence. A smallinput sample preserved on FTA can provide enough amplified DNA for multipleSNP analyses and other downstreamapplications. (See www.amershambio-sciences.com or www.genomiphi.com fordetails.) This application note shows anexample of GenomiPhi amplification ofhuman genomic DNA stored and purifiedon FTA.MethodsFollowing standard FTA protocols, freshhuman blood from a finger-stick wasspotted onto an FTA Card and allowed todry. The sample was then stored in thedark at room temperature for 5 weeks.Disks of 1.2 or 2.0 mm diameter were cutfrom the bloodstained areas with a HarrisMicro Punch. Control disks were cut fromunused areas of the card. All disks werewashed three times for 5 minutes with200 µL FTA Purification Reagent and twice for 5 minutes with 200 µL TE-1(10 mM Tris, 0.1 mM EDTA, pH 8).Amplification followed the standardGenomiPhi protocol. Each washed diskwas added to 9 µL GenomiPhi SampleBuffer. Control disks received 20 ng purified human genomic DNA beforeamplification. Samples were incubated 3 minutes at 95ºC and 3 minutes at 4ºC.10 µL GenomiPhi Reaction Buffer containing 1 µL polymerase was added to each tube. The tubes were brieflymixed by finger vortexing and incubatedovernight (about 16 hours) at 30ºC, then10 minutes at 65ºC and were finallystored at 4ºC. A 4 µL sample was removedfrom each tube for electrophoresis in 0.6%agarose containing ethidium bromide.Whatman FTA®Amplification of HumanGenomic DNA from Bloodon FTA with GenomiPhi™
Electrophoresis Gel of Amplified Human Genomic DNA from Blood on FTAResultsThe figure shows successful GenomiPhi amplification of human genomic DNA immobilizedon 1.2 mm disks cut from FTA containing humanblood (lanes 3–5). The 2.0 mm disks did not givesuccessful amplification in this experiment (lanes6–8). Added DNA was still amplified in the presence of washed FTA control disks of eithersize (lanes 9 and 10).The amplification of human DNA from bloodstored on FTA also succeeded with smaller (1.2 mm) but not larger (2.0 mm) disks in twoadditional experiments (data not shown).However, GenomiPhi did amplify DNA on largerdisks of buccal samples, while washed disks didnot inhibit amplification of control DNA. Itappears that excess DNA template on the largerblood sample disks may inhibit the process.ConclusionsHuman blood DNA preserved on FTA is a suitablesample for whole genome amplification byGenomiPhi. This approach combines the FTA benefits of collecting small original samples andstoring them safely and inexpensively at room temperature with the WGA benefits of easilyamplifying large enough quantities of DNA forthe most demanding applications.51638 11/03Additional informationon FTA products is available from Whatman.www.whatman.com or 1-800-922-0361Whatman QualityWhatman is a global leader in separations technology and is known in the scientific community for providing innovative Life Science products and solutions.Our instinct for simplification accelerates the rate of discovery, reduces costs and saves time. In order to focus on the unique needs of our customers, Whatman is organized into four business development units: Analytical Chemistry, Diagnostics, Genomics &Proteomics and Medical Devices. For more information, visit www.whatman.com.North America Whatman Inc. 9 Bridewell Place Clifton, NJ 07014 USATechnical Support: 1-800-922-0361Customer Service: 1-800-631-7290 Outside US: 973-773-5800Fax: 973-773-0168E-mail: firstname.lastname@example.orgEurope Whatman International Ltd Springfield Mill, James Whatman Way Sandling Road, MaidstoneKent ME14 2LE UKTel: + 44 (0)1622 676670Fax: + 44 (0)1622 691425E-mail: email@example.comJapan Whatman Japan KK Daiwa Ueno Building 1F 6-10 Ueno 5-chome, Taito-ku Tokyo 110-0005, JapanTel: +81 (0)3 3832 6707Fax: +81 (0)3 3832 6457E-mail: firstname.lastname@example.orgAsia Pacific Whatman Asia Pacific Pte Ltd 171 Chin Swee Road #08-01 San Centre Singapore 169877Tel: +65 6534 0138Fax: +65 6534 2166E-mail: email@example.com©Copyright, Whatman Inc., 2003.Whatman and FTA are registered trademarks of the Whatman Group.GenomiPhi is a trademark of Amersham Biosciences Ltd.Prism and BigDye are registered trademarks of Applied Biosystems.M12345678910Lane M:1 kb DNA ladderLane 1:Positive control DNA (no FTA)Lane 2:Negative control (no DNA or FTA)Lanes 3-5: Amplifying 1.2 mm sample disksLanes 6-8: Amplifying 2.0 mm sample disksLane 9:Amplifying 1.2 mm control (unspotted) disk plus positive control DNALane 10: Amplifying 2.0 mm control disk plus positive control DNALeaders in Separations Technologywww.whatman.comRecommended Protocol1) Preserve sample normally onFTA. Store in a dark, dryplace at room temperature.2) Punch disks (1.2 mm forblood) and transfer to tubes.3) Wash disks 3 x 5 minutes in 200 µL FTA Reagent.4) Rinse disks 2 x 5 minutes in 200 µL TE-1. Disks should be colorless.5) Dry disks 1 hour at room temperature (optional).6) Add 10 µL (or 9 µL for wet disks) GenomiPhi sample buffer.7) Incubate 3 minutes at 95ºC and chill on ice.8) Centrifuge briefly if necessary to collect condensate.9) Add 10 µL GenomiPhiReaction Mix containing 1 µL Phi29 polymerase andmix.10) Incubate overnight (orabout 16 hours) at 30ºC.11) Incubate 10 minutes at65ºC and store at 4ºC.00
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