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DNA extraction (Phenol chloroform Vs NaOH)

DNA extraction genotyping PCR

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5 replies to this topic

#1 Mad researcher

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Posted 27 October 2013 - 07:18 AM

Hi,

 

I have some DNA extracted by using the NaOH protocol but the yield in some is not good and all the samples are impure with 260/280 ratio ranging from 0.5-1.2

 

Therefore, i am planning to go for phenol chloroform extraction method but just wanted to know what are the advantages of this method over other methods (including kits)? I would be using the extracted DNA for genotyping and the source of tissue is from rat tails.

 

I tried running PCR's with the extracted DNA (NaOH method) but didn't get any results and i doubt it maybe because of low yield and impurities. 

 

Looking forward for some tips and advices.


Edited by Mad researcher, 27 October 2013 - 07:19 AM.

Cheers,

Mad Researcher

#2 Trof

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Posted 27 October 2013 - 12:30 PM

Didn't try phenol/chlorophorm on tails DNA, but we use it regulary for human blood. The reason is rather simple. It allows us to isolate from much more starting material than any column kits (up  to 9 ml), also it is long optimized here both with good experiences with an isolation and long-term storage.

 

But with tails.. I understand that they are trouble in general. We use Qiagen kits for mouse tail DNA (overnight digestion..) but still troublesome.

 

What helps most for genotyping from our mouse tails is simply dilute the final DNA, at least 10 times the column eluate. That's usualy enough to get a good genotyping data (even HRM based). Of course the quality or concentration wouldn't be good for other applications, but if you only want to genotype, try diluting it. Or in combination with some PCR additives - BSA (didn't try that) that can supress inhibition.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#3 Mad researcher

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Posted 27 October 2013 - 12:55 PM

Hi,

 

Could you tell me the advantage of diluting the DNA?

 

Should i be worried if the 260/280 ratio is between 0.5-1.2? Does it really matter? To me, it matters because if there are any contaminants then it may create a hindrance during the PCR?


Cheers,

Mad Researcher

#4 Trof

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Posted 27 October 2013 - 01:23 PM

Sorry, didn't mention.

Yes, the problem lies most probably in the contaminants that inhibit PCR. That's why the ratio is low and that's why your PCR doesn't work. By diluting, you dilute the inhibitors (DNA also, but you don't need that much of it, you can increase number of cycles if needed) so the reaction will work.

 

I'm just trying to tell you it's more efficient to just make the genotyping work (after all you want a plus-minus reaction, not a quantification for a genotyping, right?) by simple dilution than to try many lengths to improve the isolation, which is probably rather problematic if possible.

 

I got tail DNA from other lab. They were saying you can't genotype from those, since it's bad quality. It was, most likely. I diluted the same DNA 10 times and increased number of cycles, and the genotyping for knock in gene worked like a charm (reaction amplfies both wt and mutant-knock in, so you would have some PCR band every time, to check if it's running at all). Nothing else was needed, just to make it work. We don't need the DNA for anything else, so why bother with it's purity.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 Mad researcher

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Posted 27 October 2013 - 01:28 PM

Hi,

 

got your point. 

 

So, i would try to dilute my DNA (1:10) and also try increasing the no. of cycles after dilution.

 

Between what is your suggestion in increasing the no. of cycles i.e. +5 or +10?

 

Cheers,


Cheers,

Mad Researcher

#6 Trof

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Posted 27 October 2013 - 01:39 PM

 It depends how much patience do you have smile.png

 

I would try 10x, 100x.. 1000x? dilution in one run (to check which one will be enough if any) and increase the cycles too, to see if it amplifies at all (from a standard 30 I went to 35, but if you get nothing with 35 cycles.. well, real-time has up to 50 cycles..). You first need to know if you can amplify at all, push it to the maximum if necessary. The theoreticall PCR single-copy-limit lies somewhere near the 45 cycles in 100% efficiency if I recall right. But striking 50 at the first try.. that's probably not needed.

 

After that if you get a product, you may optimize so you won't gen nonspecific amplification and stuff.  

My standard protocol for mouse tail genotyping says now, dilute to 10 ng/ul (BUT that comes from the fact we get around 100-200 ng/ul concentration from the column kit, yours may be different, depends on concentration of DNA to concentration of inhibitors, you must find your own optimum) and 35 cycles. Perfectly specific bright bands (some less bright but still OK).


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon






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