DNA extraction with phenol chloroform and ethanol precipitation
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DNA extraction with phenol chloroform and ethanol precipitation
This protocol uses phenol/chloroform (1:1) to extract DNA samples. Phenol/chloroform extraction can be repeated for two or more times until protein interface is invisible. Purified DNA can be concentrated by ethanol precipitation. (Alam lab, University of Hawaii at Manoa)
Last update 19-Sep-2006, Rating Good of 14 votes.
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Ok protocol is fine but one should also mention the role of each and every chemical or the reagent used!!
that will help in better understanding of the protocol.
Rating: Fair
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I would like to isolate parsite DNA from stool samples. I would like to know the best protocol to use in this situation. Thanks
Rating: Excellent!
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For isolation of bacterial DNA, I recommend streaking out your sample on a plate so as to obtain individual colonies. You can then take a colony and put it in media to make lots of cells. 3mL of LB is a good start. Grow overnight, with shaking. If you don't have access to a Qiagen kit (about $1-2 per sample, smallest pack is 50 reactions) but do have a microcentrifuge and access to chemicals, you can make Qiagen buffers P1, P2, P3 (resuspension buffer with RNase, NaOH for lysis, and potassium acetate for neutralization). Follow the Qiagen protocol until you get to the bind P1+P2+P3 supernatant to column. Intstead, remove it to a new tube and add 2.5 volumes cold 100% ethanol. Spin 14K RPM for 30 minutes, dump off supernatant and you should see a little white DNA pellet. Wash with 70% ethanol, dry until edges are translucent, and resuspend in 50 uL TE buffer.
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I would like to isolate bacterial DNA from wastewater samples. I would like to know the best protocol to use in this situation. Thanks
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you can read molecular cloning by sambrook to clear your concepts about dna extraction and the role of various chemicals in it.
Rating: Good
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hello. i an doing dna extraction by phenol chloroform method . after drying the pellet i am keeping it at55c for 1hr. in TE buffer and then at 4c overnight but still pellet remain as such. on pipetting or vortexing the shearing is observed. how can i get an intact dna?
Rating: Good
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HELLO.I DO EXTRACTION DNA with phenolchloroform but i have smear can you help me immideatly thank you
Rating: Fair
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hello excuse me can you help me i do what kind of protocol for DNA extraction of ear gerbilea for good DNA.
THANKS A LOT
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Tengo una dudad si tengo en TE1x mi DNA hice diluciones de 50 microlitros a 25ng/microlitro de DNA si quiero concentrar mas mis muestras o de varias diluciones mis muestras como le puedo hacer para concentrarlas alguien tiene algun protocolo hecho en casa?o me podria explicar como? me urge saber please.
marco45@yahoo.com
Rating: Good
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hi, i am an undergraduate student and doing a practical on dna extraction, purification, quantification and restriction enzymes, can you please send me literature review about this practical.
Rating: Excellent!
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May I know the procedure given is from which source of reference? Thanks alot.
My next question is .. the DNA for purification is from what kind of extraction method? (Alkaline lysis, boiling, etc) The DNA used in purification is already suspended in buffer solutions or autoclaved water, or right after extraction before suspending in the appropriate solutions?
Thanks. Hope to hear from you.
Rating: Good
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Protocol for DNA purification
Purpose for DNA purification:
To precipitate polysaccharides and other contaminating macromoles from the DNA , and improve DNA quality.
Procedure for DNA purification:
1.Add equal volume PCI (phenol/chloroform/isoamyl alcohol, 25:24:1, from Fisher Biotech Cat: BP1752-100) to DNA hydration solution, vortex vigorously, spin 2 mins, a white interface indicates removal of polysaccharides and other contaminating macromolecules. Remove upper aqueous phase to new tube. Note: Phenol and chloroform are biohazards. Dispense in a fume hood and wear gloves.
2.Add 2.5 X volume with 100% ethanol, 10% original volume ammonium acetate (10 M)—can add 1-3 µl glycogen(Invitrogen cat. no.10814-010) here to preserve low amounts of DNA. Glycogen makes a nice visible pellet. Let mixture sit at –20ºC for ~2 hours or preferably overnight at –20ºC.
3.Spin for 20 min at 4 ºC, decant ethanol (rinse pellet with 75% ETOH if it does not move), air dry.
4.Carefully resuspend the pellets in small volumes of hydration buffer and let them dissolve at RT overnight or at 55 ºC for 2 hours.
5.Measure DNA concentration on the UV spectrophotometer. Ideal DNA concentration is 200-500 µg/ml.
Rating: Very Good
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hi, my self rohan i m doin MSc-2 microbiology ,i m doing practical on the DNA extraction but i havent standerd protocol for that, so i require it because it is important for me so plz send it to me
Rating: Excellent!
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DNA isolation from Mammalian cells and tissues
In this DNA extraction protocol, we use proteinnase K and phenol, the final DNA molecules are about 100-150 kb after several extraction steps. They are good for PCRs and Southern blotting.
Buffer:
Lysis buffer
10mM Tris-Cl (pH8.0)
0.1M EDTA(pH8.0)
0.5%(w/v)SDS
20 ug/ml DNase-free pancreatic RNase
1.Lysis cells
For adherent cell, trypsinize cell first, wash with PBS.
Add 10ml of lysis buffer for 1ml cell suspension, incubate 1 hour at 37 C.
For lysis of Tissue samples:
use Mortar and pestel, grind prechilled tissue with liquid nitroqen until sample to pulvalize sample.
Add 10 volume powder of lysis buffer in a tube and proteinase K(20mg/ml) to a final lysis buffer, incubate 3 hours at 50C. Swirl tube from time and time.
2.Treatment with phenol
Add equal volume phenol, gently mix 10 minutes and centrifuge 10 minutes at 5000g at RT.Seperate two phases.
3.Use apipette to transfer the supernanant.(aqueous phases)
repeat extraction with phenol.
4.Transfer aqueous phase to a new tube, add 0.2 volume of 10 M ammonium acetate, and add 2.5 volume 100% ethanol at RT. Mix well.See DNA fragments, centrifuge at 5000g. if not see DNA, freeze tube at -20C over night. centifuge later on.
5. wash with 70% ethanol twice
6. Disslove DNA with TE buffer.
Rating: Good
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