8 replies to this topic

[postdoc]

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julianne: been using sod acetate- isoprop precipiation with no problems all the while.

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Turtle: my suggestion would be to ethanol precipitate,

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for PCR product purification used for cloning, phenol/chloroform's classical method is ok. I think kits are unnecessary.

For cloning of PCR products, does phenol/chloroform extraction necessary? or can I just do a precipitation?

I have a basic question about DNA precipitation, sometimes isopropanol is used, sometimes ethanol is used, what is the reason for using one not the other, and what's the rule when to use one not the other?

Edited by bioforum, 29 September 2004 - 04:15 PM.

[julianne]

dear p-doc,

i'm not sure about critical works like microarray,
as the furthest i've done is seq and cloning succesfully.
{trying to plan for my 1st RT PCR run..}

i used to use phenol-chlor for my PCR product. one fine day, i decided to take a shortcut by skipping phenol-chlo and going straight to sod acetate-isoprop precipitation..
seeing that my sequencing result was still as good, i've been 'unburdened' of the phenol-chlo pain.. i used to do EST work manually.. so, imagine phenol-chlor for 2X96-well plates per day! major pain

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EtOH vs Isoprop..
i choose them according to the time and volume of my sample.

Time:
if i plan to leave my product to precipitate overnight in the -20 deg C, i use EtOH and rush home for some 'action' aka reality TV.  
isoprop i leave it for just 1.5 to 2 hours..

volume:
for EtOH: about 2.5 to 3 volume (of sample + sod acetate) added. while Isoprop: 1 volume (sample + sod ac.).
so if i have larger volume which will not take the extra 3 volumes in the Eppendorff tube, i'd choose isoprop..

but i prefer using isoprop... fast!
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hope this help.. could you pls look at my cost cutting thread in chit chat and maybe give me another 2 cents there?

THANKS

[postdoc]

Than YOU, julianne.

Leaving out phenol extraction will save lot of pain.

[tfitzwater]

Phenol extraction of PCR reactions is recommended when subsequent fill-in of the PCR product is not desirable.  Taq will stay active unless treated with proteinase K, but the phenol:chloroform step will remove most of it.  Taq survives ethanol or isopropanol precipitation.  If the PCR product is then cut with restriction enzymes, the residual Taq and dNTPs will allow the ends to become filled-in.  Then you encounter ligation failure.  

Ethanol is more heavily taxed than isopropanol.  Many labs use isopropanol because it is cheaper.

[jaregi]

The main question remains despite considerations of timing & volume:

Which is the more efficient nucleic acid precipitation method? Ethanol or 2-Propanol (Isoprop)?

Has anybody compared the amounts of precipitate?  

Edited by jaregi, 29 November 2004 - 10:40 AM.

[mikew]

Isopropanol will ppt your DNA faster, but will also give you a higher amt. of salt ppt.
Ethanol takes longer. My experience, but I cannot absolutely say it is so, is that a 1-2 hr. etoh. ppt gives a better yield than a quick isopropanol.
For ligating, no phenol:chlor is necessary.

[izeman]

I use ethanol instead of Isopropanol,but instead if the time consuming -80 incubation...i just pop it into liquid nitrogen and instant pptation can be seen.So incase you need to use ethanol and need quicker runs...this would be helpful

[jaregi]

Hi MikeW! Thanks for the reply. I think salt co-precip depends on temperature. One actually precipitate less salt with 2PrOH (isoprop) precipitation at room temp. compared to EtOH precipitation at -20/-80degC.

This seems to be the biggest advantage of 2PrOH precipitation. You could probably increase the DNA yield there by also putting it at low temperature, but then you would get more salt.

I still wonder if you compare DNA yield for RT 2PrOH vs. cold EtOH, which one gives more DNA??

[jaregi]

I actually ran test on EtOH versus 2PrOH (isopropanol) precipitation efficiency.

16 minipreps divided into 2 equal volumes and then precipitate either with EtOH or 2PrOH. Note that to speed up the ethanol precipitation I dipped it into liquid nitrogen instead of putting it at -80°C for 30min to 1hours like some protocols recommend.

The result was very clear: ethanol precipitated on average 60% more DNA. I would ethanol precipitation is not as good if you precipitate at -80°C. Btw, I also cooled the 2-propanol mixes to 4°C since I was working in the cold room.