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Ethanol precipitation vs Clean up kit? - which will yield more (Oct/19/2006 )

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Hi,
I have to do sequential digestion of a PCR product, so I need to precipitate DNA after each digestion. However, we do not have a speed vac and thus have to leave DNA overnight to dry, this way one cloning experiment takes forever.
I also have tons of Qiagen PCR purification kits lying around and I looked up the qiagen kit usability chart, it says I could use the PCR purification kit to clean up a restriction digest as well.

Please give your ideas on whether this would be a wise idea?

Thanks a bunch smile.gif

-nifT-

i would choose ethanol ppt. over a kit.

-scolix-

QUOTE (nifT @ Oct 19 2006, 01:05 PM)
Hi,
I have to do sequential digestion of a PCR product, so I need to precipitate DNA after each digestion. However, we do not have a speed vac and thus have to leave DNA overnight to dry, this way one cloning experiment takes forever.
I also have tons of Qiagen PCR purification kits lying around and I looked up the qiagen kit usability chart, it says I could use the PCR purification kit to clean up a restriction digest as well.

Please give your ideas on whether this would be a wise idea?

Thanks a bunch smile.gif



we were using the Qiagen kits to purify gel products and couldn't figure out forever why our ligations wouldn't work. switched to old-school phenol/chloroform extraction and the ligation worked like a charm right away.

-pipet_overuse-

I am not sure about kits... I only use gel extraction kit.

However for EtOH percipitation you do not need to leave the tube to dry over night.
After the percipitation, spin, 70% EtOH wash, spin...and removal of the 70% EtOH wash...

-you can dry your DNA in a hybridisation incubator (2 mins is usually enough and dry no more then 3mins)

-dry on a heating block set at 65C (no more then 5mins)

-or you can do a quick 1min spin, then remove any remainder supernatant. The tube should then dry in about 5 to 10 mins at room temperature.

-perneseblue-

[quote]I am not sure about kits... I only use gel extraction kit.

However for EtOH percipitation you do not need to leave the tube to dry over night.
After the percipitation, spin, 70% EtOH wash, spin...and removal of the 70% EtOH wash...

-you can dry your DNA in a hybridisation incubator (2 mins is usually enough and dry no more then 3mins)

-dry on a heating block set at 65C (no more then 5mins)

-or you can do a quick 1min spin, then remove any remainder supernatant. The tube should then dry in about 5 to 10 mins at room temperature.[quote]

i simply remove ethanol and let dry around 10 minutes at room temp or until no liquid is seen in the tube anymore.

here in Japan there is something called "ethachinmate", don;t try to understand the name it is a mixture of japanese and english laugh.gif . you put 1 ul of it with your ethanol and no matter how little of DNA you have you will still be able to see the white precipitate. i just love it wub.gif.

-Kathy-

Hi,

I prefer to gel isolate and purify. To PCR purify is ok though, just wash more than once with wash solution. To make sure no residual ethanol, can heat at 50degreeC, after adding TE or water, for 5' to 10' before elution step. That'll evaporate any EtOH and increase yield.

Both kit and phenol-chloroform + EtOH methods are ok for cloning steps. I prefer the former, less messy and smelly.

Bests

-I love MSGs!-

QUOTE (Kathy @ Oct 20 2006, 03:19 AM)
i simply remove ethanol and let dry around 10 minutes at room temp or until no liquid is seen in the tube anymore.

here in Japan there is something called "ethachinmate", don;t try to understand the name it is a mixture of japanese and english laugh.gif . you put 1 ul of it with your ethanol and no matter how little of DNA you have you will still be able to see the white precipitate. i just love it wub.gif.


My guess would be "ethachinmate" is dextran sulphate. Great stuff to help percipitate DNA. The natural stuff is white, though it is possible to purchase dyed dextran, coloured either blue, red and green.

-perneseblue-

I am surprised by how many people dislike the kits. I use them all the time and have pretty good success with ligations (blunt and sticky). Horses for courses I guess...

-ML1975-

QUOTE (perneseblue @ Oct 20 2006, 09:05 AM)
QUOTE (Kathy @ Oct 20 2006, 03:19 AM)


i simply remove ethanol and let dry around 10 minutes at room temp or until no liquid is seen in the tube anymore.

here in Japan there is something called "ethachinmate", don;t try to understand the name it is a mixture of japanese and english laugh.gif . you put 1 ul of it with your ethanol and no matter how little of DNA you have you will still be able to see the white precipitate. i just love it wub.gif.


My guess would be "ethachinmate" is dextran sulphate. Great stuff to help percipitate DNA. The natural stuff is white, though it is possible to purchase dyed dextran, coloured either blue, red and green.

I found no difference between kit (qiaqick) and do-it-yourself, I use the faster one, normally the kit.
For drying the ethanol percipitates we use a Speed Vac.
To help precipitating the DNA, Glycogen also helps a lot.

-hobglobin-

QUOTE (hobglobin @ Oct 20 2006, 12:18 PM)
QUOTE (perneseblue @ Oct 20 2006, 09:05 AM)

QUOTE (Kathy @ Oct 20 2006, 03:19 AM)


i simply remove ethanol and let dry around 10 minutes at room temp or until no liquid is seen in the tube anymore.

here in Japan there is something called "ethachinmate", don;t try to understand the name it is a mixture of japanese and english laugh.gif . you put 1 ul of it with your ethanol and no matter how little of DNA you have you will still be able to see the white precipitate. i just love it wub.gif.


My guess would be "ethachinmate" is dextran sulphate. Great stuff to help percipitate DNA. The natural stuff is white, though it is possible to purchase dyed dextran, coloured either blue, red and green.

I found no difference between kit (qiaqick) and do-it-yourself, I use the faster one, normally the kit.
For drying the ethanol percipitates we use a Speed Vac.
To help precipitating the DNA, Glycogen also helps a lot.


glycogen aka dextrin... glycogen from bacterial origin.

-perneseblue-

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