Ethanol vs Isopropanol for DNA precipitation - (Sep/23/2004 )

to my knowledge and exprerience, phenol/chloroform extraction is no need for cloning of PCR product. To rule out the enzyme in it, kit from some company uses column. But it is the same when directly pricipitation.
for DNA precipitation, the function of isopropanol and ethanol is the same. but isopropanol works better. When your DNA sequence is larger than 6kb, isopropanol is recommented.

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I want to know why we keep the DNA samples in cold temp after adding ethanol or isopropanol ?

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EtOH can recover small nucleic acid ( even 10bp(nt)), while isopropanol not.

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Well it does matter even if u keep the DNA sapmles at room temerature

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Can anyone tell me how do I increase the isopropyl precipitation of DNA yield?

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For DNA or RNA to be recovered by low speed centrifugation, the molecules have to collapse on each other to make a particle large enough to be drawn out of suspension by centrifugation. If the molecules did not "grab"onto each other in the presence of alcohol, you would have to use centrifugal fields of 100s of thousands of G force to pull the molecules out to pellet. The cold temperature accelerates the flocculation process. With Isopropanol, for larger DNA that is not necessary since the solubility of the DNA is not as great in isopropanol than ethanol. To get DNA to fall out of solution in ethanol you have to be at 75% concentration and with salt concentration approaching 0.5M, with isopropanol the salt concentration at 0.5 M though at 35% Isopropanol. Now smaller nucleic acids are just more soluble in either alcohol, and if one wanted to figure it out, Ethanol would fractionate small from big pieces only at different concentrations. So if you use more Isopropanol, you will get smaller pieces to ppt as well. However, for PCR, to remove the primers, and nucleotides, and your product is greater than 100bp, and you can suffer some loss of product, since you have plenty, I use Isopropanol at 0.6vol or 35%. About the Taq surviving, after electrophoresis (it would move to the opposite pole) I add a formamide (20%) in the presence of the acetate salt at 0.5M, heat to 50C for a 5 to 10 minutes, then precipitate with Isopropanol. Always wash your pellet with 50% to 75% Ethanol with a 0.15 NaCl, assumming >100bp and more than a 0.1ug, to remove any residual anything that is on the tube etc, which came down with the Isopropanol. Do it twice to please you. If you do not trust this process, try it by adding Taq and see how much survives.

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Hi All
I am doing DNA isolation by phenol chloroform ext method. then i am precipitating by ethanol overnight. i get a reasonable good DNA. then i am dissolving in TE, but the sample is viscous and difficult to load into gels. how to reduce viscosity, so that sample loads in wells easily.
i hope any one of you may help/ suggest at your earliest.
regards
saty

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do the same at lower temperatures and for long duration...say overnight.

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also because at low temperatures brownian movement is less....surf the forum and u will get detailed reason....i saw something yesterday in Molecular Biology forum.

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Hi all

Can any one tell that, can we take break in protocol of DNA isolation/purification/extraction.....by freezing the samples in between steps. will it going to harm the yield/beauty of DNA isolated.

also tell me some tips to beutify gels.....to avoide smear...etc.

i would be very thankful if any one rushes his remarks or refer me some site/link/person.

regards
saty

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