I want to precipitate genomic DNA extracted by PCI (Phenol:Chloroform:isoamylalcohol, 25:24:1 ratio) method and for that I mistakenly add sodium-acetate pH 4.2 (instead of pH 5.2) + 100% ethanol, followed by -80C incubation for 10 mins but when I centrifuged it 13000rpm for 12mins, pellet was not formed/seen. So please suggest me how to precipitate gDNA from this solution.
The pH of sodium acetate is not THAT critical. See
https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/sodium-acetate
"Sodium acetate is the standard saline solution used by experimenters. The concentration of the DNA solution (i.e., final concentration) before the addition of any alcohol should be 0.3 M. To accomplish this, the experimenter uses a 3 M sodium acetate solution at a pH of 4.8 to 5.2."
It could be simply that the quantity of DNA you precipitated is so small that you can't see it in the tube. In your current situation, proceed by centrifuging the tube one more time at 15000 rpm for 15 min, then carefully remove supernatant (try not to touch the tube walls and bottom), wash by 70% ethanol and try to resuspend the invisible DNA (let's think it's there). Resuspend in REALLY small quantity of water carefully by pipetting and trying to "gather" stuff from walls and bottom, and measure concentration. You will get an answer whether you have the DNA there or not.
Subhash Ph.D., the point raised by Yulia Panina is noteworthy. Nonetheless, in your case I wonder whether using a solution under the pKa of acetic acid (4.76) would be that harmless to salt-based precipitation. I may be wrong but I guess that is why you see pH >4.8 in every single protocol. Under this assumption salting out sounds reasonable.
Perhaps you could save your DNA by diluting and precipitating it with isopropanol -if it worths the try.
Please let us know.
Best,
D
Bring your sample at room temperature and add 1-2 μl glycogen (20μg/μl); mix and add eventually a little bit more ethanol (the ethanol concentration you already have in your sample could be not sufficient); incubate 30 or more min at -80°C; centrifugate 10 -15 min or more at 12 000g at 4-8°C; (at this stage you should observe a nice pellet); through out the supernatant and perform two washes with 75% cold ethanol.
thanks to Yulia Panina mam and Dimitar Angelov sir, I have used yours points to precipitate gDNA..and I got it (NanoDrop spectrophotometry reading & peaks are fine).
but one problem is come that DNA is showing smear on agarose gel (1%). I have isolated gDNA from 7 samples and when I run PCR on these isolated gDNA, only one sample showing amplification, while common master mix was prepared for all samples (only template was vary).(tried twice)
so please suggest me the reasons why i'm not getting amplification in other samples? while similar procedure was performed for all samples.
Subhash Ph.D. thanks for letting know the result! It's good to know it worked.
About the PCR, I didn't quite understand the difference between 7 samples. If PCR worked on only one of them, maybe it means that the target was present in only one of them? Are the 7 samples identical or something is different between them (before gDNA extraction)?
Yulia Panina mam, there are nothing in difference between 7 samples. same procedure was performed for all samples and target is present in all samples (samples are from one bovine species i.e. Bos indicus' sperm cells).
Your questions show you are a beginner. If your boss is not able to guide you and provide advices/help during all steps change the boss.
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