you can use Trizol for DNA & RNA extraction, many protocols u will get online regarding TRIZOL.
i searched but I could'nt zero on any one...can someone help please, I really need it
Dear Himanshi, I found a protocol that might be of use to you. Please see this link: http://cancer.ucsf.edu/_docs/cores/array/protocols/dna_cell_culture.pdf
I hope it helps!
Best wishes,
You can try these links:
https://www.researchgate.net/post/DNA_extraction_from_TRIZOL_sample or
http://shared-resources.dhvi.duke.edu/files/DNA_RNA_Isolation_TRIZOL.pdf
or
http://www.mrcgene.com/tri.htm
In principle you can use the classic Proteinase K digestion and phenol chloroform extraction with subsequent DNA precipitation. I have done this several times and it works for cell lines. Another many times used Kit is the QAmp DNA Mini Kit from Qiagen. DNA from the Kit has not to be precipitated afterwards. From the columns the DNA is clean enough.
you can put 1 ml of tryzol for a 6 mm dish wait 5 minutes and pipette, then you can store at -80°C and find on web a protocol to extract DNA or RNA
This is a protocol for DNA extraction from a large petri dish.
cells were washed twice with 25 ml of ice-cold PBS, and lysed in 9 ml lysis buffer (400 mM Tris-HCl, 60 mM EDTA, 150 mM NaCl, 1% w/v SDS, pH 8.0). DNase-free RNase A was added to a final concentration of 10 mg/ml, and incubated at 37°C for 1 h. Three millilitres of 5 M sodium perchlorate were added and the mixture was mixed by inversion at room temperature for 20 min and then incubated at 65°C for 30 min. The samples were then cooled in an ambient water bath for 5 min and extracted twice with 15 ml of chloroform. After centrifugation at 2500 g for 20 min, 10 ml of the aqueous supernatant was removed and DNA precipitated by the addition of 25 ml absolute ethanol at −20°C. The genomic DNA was then washed twice in 70% ethanol and dried. DNA was wetted with 20microlitres of ddH2O (taken from 500microlitres) for 10 minutes. The remainder of the ddH2O was added, and the DNA left to dissolve over night at 4C.
The genomic DNA can be quite a pain to work with if you do not dissolve it properly, but this is a really cheap and easy way to make genomic DNA. It can also be scaled down to an eppendorf tube scale by using less cells, for example 500 microlitres of lysis buffer for one well of a 6 well plate, everything else being in the same ratio and you can use a bench-top microfuge to spin the chloroform extractions. The DNA normally precipitates OK, but if you have poor yield add sodium acetate before the ethanol precipitation step.
Paper title: A novel disintegrin protein from Naja naja venom induces cytotoxicity and
apoptosis in human cancer cell lines in vitro
Please refer the following article to extract the DNA from cancer cells.
Paper Title: A novel disintegrin protein from Naja naja venom induces cytotoxicity and
apoptosis in human cancer cell lines in vitro
Qiagen has a variety of easy-to-do, fast and reliable kits for the extraction of DNA; the resultant DNA quality is exceptional.
DNA extraction and purification is very simple nowadays. there are a lot available kits. Cheaper and with high efficiency: TRIZOL. Good also for any other tissues.
Could you specify what you are planning to do? Its a big difference, if you want to do genotyping, southern blotting/hybridization, or PCR. For our mouse-genotyping (PCR) we use the Qiagen DNeasy -Kit, which is not cheap, but works relably for lazy people like us. if you are on a budget, I can highly recommend some of the glassmilk-protocols. They cost close to nothing and give good yield of reasonably clean DNA, personally I try to stay away from TRIZOL (which is essentially the "acid phenol method" by Chomzynski et al, originally used for RNA (not DNA) isolation, however, it lets you "rescue" the DNA afterwards. I would rather use a method specifically for DNA. For a glassmlk -protocol go to: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007750
or:
http://herpesvirus.tripod.com/research/protoDNA.htm
Trizol tends to leave traces of Phenol, which can kill enzymatic reactions, and is a nasty, carcinogenic environmental hazard. Therefore I like to avoid it as good as I can. Except for RNA isolation, I do not use Phenol at all anymore. But maybe I'm overly sensitive, use of a chemical hood and proper precautions with the waste certainly allow you to do all the Phenol work you want to do in a safe and responsible way.
Due to my experience with commercial kits I can recommend QIAGEN DNA mini kit, I have used them for different cell lines as well as primary cells in a very successful manner. I was working with different cancer cell lines as well as primary cells (endothelial cells as well as ovarian and endometrium cancer cells) and I never faced any problems. The kits are simple in handling and you get good results.
If the goal is to have DNA of the "transfection-grade" quality I would not use any kit that renders DNA at A260/A280 to be below 1.8, which would be the "TRI" from Sigma (with the Sigma "TRI" you would also have both RNA and protein contaminants).
Qiagen allows to isolate and purify the DNA at close to 2.0 (protein and RNA free). If the aim is to have pure DNA, I would also recommend using those kits working on gravity-flow through the column. Promega's kits where you would use the vacuum generally render lesser quality of DNA.
A hint: Most companies would provide a "test-sample" of either of their kits for you to try it out free of charge. Sometimes this is a cost-saving resolution:)) Good luck!
Dear Himanshi, ¿Nuclear DNA or mitochondrial DNA? ¿what do you need?
Regards Jose Ramos Vivas
If your aim is to just do PCR then you can use "DirectPCR (cell)" lysis buffer from Viagen Biotech (Cat # 302-C). We have used this reagent for variety of cancers cells including glioblsatoma. 0.5-2uL of this crude lysate can be directly used for a PCR reaction!
If you need pure DNA then the best way is to isolate genomic DNA using a buffer containing Tris-HCl (100 mM), NaCl (200 mM), EDTA (5 mM) and SDS (0.2%). Precipitate DNA using 5M NaCl, wash with 70% Ethanol, air-dry and solubilize in TE.
Qiagen also has a kit "DNeasy Kit" for genomic DNA isolation from human cells.
:)
hi..may be this article titled"quantyfying the amount of DNA by using the mouse and human cancer cells in a good length".can be useful for your research..and see the attachment.this article was published by john hopkins school of medicine.
OK, what is your next downstrean application ? Cloning, PCR, verification of DNA damage, DNA quantitation ? This is important to choose the method...
OK, then, follow this protocol: is very easy to observe DNA damage.
Ioannou YA, and Chen FW, Quantitation of DNA fragmentation in apoptosis,
622 Nucleic Acids Res 24 (1996) 992-993. FREE ON PUBMED Is one of the easiest protocol to do that. we use it in our recent paper. Microbes Infect. 2013 Jun;15(6-7):480-90 with macrophages, but we do the same for VERO, A549 or COS-2 cells with excellent results. tell me if you need our pdf.
J774A.1 cells were seed in 6-well tissue culture
plates. DNA fragmentation of infected macrophages was measured by following a
modified version of a protocol described elsewhere [16]. Briefly, to exclude the
possibility of leakage of fragmented low-molecular-weight DNA from cells during
washing, the DNA was isolated from the whole cell culture volume in each well (2ml)
and the remaining cells still attached to the wells. At 2 h post-infection, cells from 2
wells were lysed in 600 l of lysis buffer (0.6% SDS, 10mM EDTA, 10mM Tris, and
20g/ml RNase A, pH8) for 1h at 37ºC. Sixty microliters of 5 M NaCl were then added,
and the preparation was incubated for 1 h on ice and centrifuged for 30 min at 13.000
rpm and 4ºC using a microcentrifuge. The supernatant containing the DNA was
extracted with Phenol-chloroform-isoamyl alcohol (25:24:1) (Invitrogen), and low
molecular-weight DNA was precipitated with cold 100% ethanol. Precipitates were
pelleted by centrifugation (13,000 rpm, 15 min, 4ºC), washed with 70% ethanol, and
air-dried. Samples (2 ug DNA per lane) were separated by electrophoresis on a 1.2%
gel and visualized by SYBRSafe staining. As a positive control for the technique
used, DNA isolated from macrophages cultured for 24 h in the absence of serum was used
tell me if you need our pdf.
Jose
Yes i would like to see the pdf, if possible, but this is also good to follow! Thanx a lot!
God bless!
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