Ethanol precipitation of DNA - (Jun/05/2015 )
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 :)
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!
Mad Researcher on Fri Jun 5 13:29:47 2015 said:
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
pito on Fri Jun 5 17:02:15 2015 said:
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!
Mad Researcher on Fri Jun 5 13:29:47 2015 said:
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
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?
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.
mdfenko on Fri Jun 5 17:54:39 2015 said:
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?
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?
mdfenko on Fri Jun 5 18:04:32 2015 said:
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?
it shouldn't be too long. but, don't get discouraged if it's difficult to suspend, just warm it to assist solvation.
mdfenko on Fri Jun 5 18:13:59 2015 said:
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?
add water and then heat it