Ethanol Precipitaion - (Feb/10/2009 )
pcrman on Feb 10 2009, 10:30 AM said:
First, what kit did you use for gel purification? You can use Zymo gel purification kit which gives you very concentrated DNA.
If you just want to concentrate your DNA from your gel purification, you can go ahead doing ethanol precipitation.
If your DNA is still in the gel, you need first to get the DNA out of it by spinning (puncture a hole in the bottom of a 0.5 ml tube using a needle) or some other ways and then precipitate your DNA.
If you just want to concentrate your DNA from your gel purification, you can go ahead doing ethanol precipitation.
If your DNA is still in the gel, you need first to get the DNA out of it by spinning (puncture a hole in the bottom of a 0.5 ml tube using a needle) or some other ways and then precipitate your DNA.
hi there. about the ethanol precipitation part,
is it just wash the DNA in ethanol for few times ? or is there any particular standard protocol for that ?
thanks
-tyrael-
NiceButDim on Feb 11 2009, 04:07 AM said:
Hey guys, just a very quick one if I may
I'm double digestion my vector (working well)
Purifying it on a gel
Excising the appropriate band (get lovely bands)
Normally the next step would to be do a gel extraction but for some reason the kits are giving me very very low yields (almost nothing). Have decided to maybe go back to basics and try a phenol-chloroform extraction and see what the problem is.
My question to you is, since my sample is purely DNA, can I skip the phenol-chloroform part and simply jump straight to the ethanol precipitation? Do I just dissolve my excised band in water and proceed with the precipitation or do I need to dissolve my sample in a particular buffer to avoid interference from the agarose?
Many many thanks for anyone able to help, really very much appreciate it guys.
I'm double digestion my vector (working well)
Purifying it on a gel
Excising the appropriate band (get lovely bands)
Normally the next step would to be do a gel extraction but for some reason the kits are giving me very very low yields (almost nothing). Have decided to maybe go back to basics and try a phenol-chloroform extraction and see what the problem is.
My question to you is, since my sample is purely DNA, can I skip the phenol-chloroform part and simply jump straight to the ethanol precipitation? Do I just dissolve my excised band in water and proceed with the precipitation or do I need to dissolve my sample in a particular buffer to avoid interference from the agarose?
Many many thanks for anyone able to help, really very much appreciate it guys.
Something else to think about:
How large is the fragment you remove by digestion? If it's under ~100 bp, you can EtOH precipitate using ammonium acetate instead of sodium acetate (you also have to change the volumes slightly, use 1/2 volume 7.5 M NH4. acetate and 2 vol EtOH).
If you're going on to clone something into the vector, treat the precipitated vector with phosphatase before cloning; this will make sure that any pieces of the digested DNA that are hanging around cannot clone.
Wear your lucky socks, jocks, hair band or whatever works for you!
-swanny-