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Ethanol precipitation of DNA

ethanol precipitation DNA nucleic acid

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21 replies to this topic

#1 Mad Researcher

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Posted 05 June 2015 - 05:29 AM

Hello,

 

I re-precipitated my DNA today with Sodium Acetate. I followed a protocol that i found online and use 1/10th volume of 3M sodium acetate . Now, i saw another protocol where it says the final concentration of sodium acetate should be 0.3 M which i didn't. I got yield of DNA as negative values i.e -200.2 and -18.2 ng/ul.

 

My question is: should i have changed the concentration of sodium acetate to 0.3M and used. Why did i get negative values although weight can't be negative.

 

What protocol do you suggest?

 

Thanks :)


Cheers,

Mad Researcher

#2 pito

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Posted 05 June 2015 - 09:02 AM

Its the same...

1/10 of 3M will be 0,3 in the end..

(before you add the ethanol! I guess thats where the confusion came from?!)

 

Some people do not even add the sodium acetate...

 

The problem is not the sodium acetate.

 

The negative values? Hard to tell.. perhaps your sample got dirty somehow? Is the nanodrop ok?

Negative weight can not be the case, but its not like that... You do not really measure "weight" .... its a bit more complicated!

 

Hello,

 

I re-precipitated my DNA today with Sodium Acetate. I followed a protocol that i found online and use 1/10th volume of 3M sodium acetate . Now, i saw another protocol where it says the final concentration of sodium acetate should be 0.3 M which i didn't. I got yield of DNA as negative values i.e -200.2 and -18.2 ng/ul.

 

My question is: should i have changed the concentration of sodium acetate to 0.3M and used. Why did i get negative values although weight can't be negative.

 

What protocol do you suggest?

 

Thanks smile.png

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Mad Researcher

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Posted 05 June 2015 - 09:26 AM

Its the same...

1/10 of 3M will be 0,3 in the end..

(before you add the ethanol! I guess thats where the confusion came from?!)

 

Some people do not even add the sodium acetate...

 

The problem is not the sodium acetate.

 

The negative values? Hard to tell.. perhaps your sample got dirty somehow? Is the nanodrop ok?

Negative weight can not be the case, but its not like that... You do not really measure "weight" .... its a bit more complicated!

 

Hello,

 

I re-precipitated my DNA today with Sodium Acetate. I followed a protocol that i found online and use 1/10th volume of 3M sodium acetate . Now, i saw another protocol where it says the final concentration of sodium acetate should be 0.3 M which i didn't. I got yield of DNA as negative values i.e -200.2 and -18.2 ng/ul.

 

My question is: should i have changed the concentration of sodium acetate to 0.3M and used. Why did i get negative values although weight can't be negative.

 

What protocol do you suggest?

 

Thanks smile.png

 

Yes, the confusion was with the ethanol.

 

The nanodrop is OK. i calibrated it last week and i also measured some other nucleic acid after this and they were fine. What else could be wrong. Although the 260/280 ratio had improved. What else could be wrong?


Cheers,

Mad Researcher

#4 mdfenko

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Posted 05 June 2015 - 09:54 AM

are you suspending the dried pellet in the same solvent that you use to blank the spec?

 

if there is any ethanolic solvent left over after precipitating and pelleting the dna then you will get a different (presumably lower) reading on the spec.


Edited by mdfenko, 05 June 2015 - 09:55 AM.

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#5 Mad Researcher

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Posted 05 June 2015 - 10:00 AM

are you suspending the dried pellet in the same solvent that you use to blank the spec?

 

if there is any ethanolic solvent left over after precipitating and pelleting the dna then you will get a different (presumably lower) reading on the spec.

Yes, i am suspending the dried pellet in water and using water to blank the spec.

 

I left to dry the ethanol for 1.30 hr. Maybe i should leave it overnight. There may be a bit of ethanol left which didn't dry. Will there be a consequence if i leave the pellet to dry for 2 days or overnight (over the weekend)? Also, in one of the tube (200 ng/ul) there was a very tiny tiny pellet and the other one (18.2 ng/ul) had a pellet which was visible. Does this make a difference?


Cheers,

Mad Researcher

#6 mdfenko

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Posted 05 June 2015 - 10:04 AM

if it gets too dry it may be too difficult to resuspend (you may have to warm it).

 

did you add glycogen to aid the pelleting?


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#7 Mad Researcher

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Posted 05 June 2015 - 10:07 AM

if it gets too dry it may be too difficult to resuspend (you may have to warm it).

 

did you add glycogen to aid the pelleting?

No, i didn't add glycogen (the protocol never mentioned it).

so, if i keep it overnight i guess it would be fine? or do you think it would be too long?


Cheers,

Mad Researcher

#8 mdfenko

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Posted 05 June 2015 - 10:13 AM

it shouldn't be too long. but, don't get discouraged if it's difficult to suspend, just warm it to assist solvation.


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#9 Mad Researcher

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Posted 05 June 2015 - 10:17 AM

it shouldn't be too long. but, don't get discouraged if it's difficult to suspend, just warm it to assist solvation.

So, how should i warm it? Should i add water and heat it or just heat it in the heating block at around 55 degrees?


Cheers,

Mad Researcher

#10 mdfenko

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Posted 05 June 2015 - 10:22 AM

add water and then heat it


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#11 Mad Researcher

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Posted 05 June 2015 - 10:24 AM

Thanks a lot :)

 

So, to sum it up:

I will keep it overnight for drying.

Next, if the pellet is really dry or not visible then will add around 50 ul water and heat at 55 degrees and check the absorbance.


Cheers,

Mad Researcher

#12 pito

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Posted 05 June 2015 - 10:40 AM

 

are you suspending the dried pellet in the same solvent that you use to blank the spec?

 

if there is any ethanolic solvent left over after precipitating and pelleting the dna then you will get a different (presumably lower) reading on the spec.

Yes, i am suspending the dried pellet in water and using water to blank the spec.

 

I left to dry the ethanol for 1.30 hr. Maybe i should leave it overnight. There may be a bit of ethanol left which didn't dry. Will there be a consequence if i leave the pellet to dry for 2 days or overnight (over the weekend)? Also, in one of the tube (200 ng/ul) there was a very tiny tiny pellet and the other one (18.2 ng/ul) had a pellet which was visible. Does this make a difference?

 

 

No no do not let it dry overnight or over the weekend!

You do not want a completely dried out pellet (it will be hard to dissolve again).

 

Its hard to tell what went wrong, many things, most likely some sort of contaminant.. like the ethanol or something else. Or maybe your blank was dirty (some dirt got in?)

The pellet: pure dna is not visible if you spin it down and do an ethanol precipitation! The cleaner the DNA the less you see it!

Its actually just dirt you see!

 


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#13 Mad Researcher

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Posted 05 June 2015 - 10:42 AM

 

 

are you suspending the dried pellet in the same solvent that you use to blank the spec?

 

if there is any ethanolic solvent left over after precipitating and pelleting the dna then you will get a different (presumably lower) reading on the spec.

Yes, i am suspending the dried pellet in water and using water to blank the spec.

 

I left to dry the ethanol for 1.30 hr. Maybe i should leave it overnight. There may be a bit of ethanol left which didn't dry. Will there be a consequence if i leave the pellet to dry for 2 days or overnight (over the weekend)? Also, in one of the tube (200 ng/ul) there was a very tiny tiny pellet and the other one (18.2 ng/ul) had a pellet which was visible. Does this make a difference?

 

 

No no do not let it dry overnight or over the weekend!

You do not want a completely dried out pellet (it will be hard to dissolve again).

 

Its hard to tell what went wrong, many things, most likely some sort of contaminant.. like the ethanol or something else. Or maybe your blank was dirty (some dirt got in?)

The pellet: pure dna is not visible if you spin it down and do an ethanol precipitation! The cleaner the DNA the less you see it!

Its actually just dirt you see!

 

 

Hahaha... Ok then change of plan.

I will keep it for drying for approx 2-3 hours and then resuspend it again?

Should i do the resuspension in TE buffer instead of water? I am sure it wont make any difference apart from better stability and long term storage.


Cheers,

Mad Researcher

#14 pito

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Posted 05 June 2015 - 10:45 AM

no need to dry it overnight!

Really no need!


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#15 pito

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Posted 05 June 2015 - 10:47 AM

 

 

 

are you suspending the dried pellet in the same solvent that you use to blank the spec?

 

if there is any ethanolic solvent left over after precipitating and pelleting the dna then you will get a different (presumably lower) reading on the spec.

Yes, i am suspending the dried pellet in water and using water to blank the spec.

 

I left to dry the ethanol for 1.30 hr. Maybe i should leave it overnight. There may be a bit of ethanol left which didn't dry. Will there be a consequence if i leave the pellet to dry for 2 days or overnight (over the weekend)? Also, in one of the tube (200 ng/ul) there was a very tiny tiny pellet and the other one (18.2 ng/ul) had a pellet which was visible. Does this make a difference?

 

 

No no do not let it dry overnight or over the weekend!

You do not want a completely dried out pellet (it will be hard to dissolve again).

 

Its hard to tell what went wrong, many things, most likely some sort of contaminant.. like the ethanol or something else. Or maybe your blank was dirty (some dirt got in?)

The pellet: pure dna is not visible if you spin it down and do an ethanol precipitation! The cleaner the DNA the less you see it!

Its actually just dirt you see!

 

 

Hahaha... Ok then change of plan.

I will keep it for drying for approx 2-3 hours and then resuspend it again?

Should i do the resuspension in TE buffer instead of water? I am sure it wont make any difference apart from better stability and long term storage.

 

 

2-3 hours, thats already too long...

30 minutes should do the trick normally!

You can pour of the liquid and remove the rest with a pipette tip (10µl) and then just dry it.

 

You can resuspend it in TE or water.. makes no real difference.. for long term storage or if you open/close the tube a lot during your work perhaps TE is better.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






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