Ethanol precipitation of DNA - (Jun/05/2015 )
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.
Mad Researcher on Fri Jun 5 18:00:50 2015 said:
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?
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!
pito on Fri Jun 5 18:40:07 2015 said:
Mad Researcher on Fri Jun 5 18:00:50 2015 said:
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?
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.
no need to dry it overnight!
Really no need!
Mad Researcher on Fri Jun 5 18:42:09 2015 said:
pito on Fri Jun 5 18:40:07 2015 said:
Mad Researcher on Fri Jun 5 18:00:50 2015 said:
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?
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.
btw: if you resuspend it, let it sit for a while, do not add the water/TE and measure the concentration right away, wait a few minutes, heat maybe, just let it "sit" a while then measure...
Normally its pretty straightforward.
I guess you just had some bad luck with this particular sample that gave a weird result.
i did say it would be difficult to dissolve if over dried.
you can, maybe should, use te but it depends on what you plan to do with it next. you can also use tris without the edta.
pito is correct, the pure dna pellet is virtually invisible. that's one of the reasons to add glycogen.
Thanks, i will let you know perhaps on Monday what happened.
So, after the ethanol precipitation the 260/280 and 260/230 ratio should inc? will it affect the yield also?
Mad Researcher on Fri Jun 5 18:54:42 2015 said:
Thanks, i will let you know perhaps on Monday what happened.
So, after the ethanol precipitation the 260/280 and 260/230 ratio should inc? will it affect the yield also?
should what? inc?
THe ratios will be a bit different. I am not sure, but I thinks its the 260/280 ratio that is going to be a bit higher..
One of them is a bit higher !
THe other one: a little bit higher, rather 2,1-2,2 than 1,8 , If I remember correctly.
The yield? Not sure what you mean... You will lose some DNA of course...
yes. you will end up with less than you started.