ethanol precipitation - (Jul/22/2009 )
Hi guys
I am wanting to perform ethanol precipitation on a solution of plasmid which I had extracted using the qiagen spin mini prep in order to try and increase the plasmid concentration. I was researching on the internet and there are many protocols out there for ethanol precipiration but I was wondering what protocols most people use??? ie the most efficient.
Thank you so much for helping me out.
Htet
Ms new scientist on Jul 22 2009, 09:18 PM said:
I am wanting to perform ethanol precipitation on a solution of plasmid which I had extracted using the qiagen spin mini prep in order to try and increase the plasmid concentration. I was researching on the internet and there are many protocols out there for ethanol precipiration but I was wondering what protocols most people use??? ie the most efficient.
Thank you so much for helping me out.
Htet
Add 10% volume of 3M sodium acetate, then 1.5, 2.0, or 2.5 volumes of 100% EtOH. Mix well, but gently. I've always incubated at -20C for an hour, but some I know have done it at room temp and swear it works just as well. Pellet the DNA, wash with 70% EtOH, allow pellet to air dry (but don't overdry), and resuspend in TE, tris, water, whatever. I've never done this for plasmid preps, but it works well for genomic DNA.
Another option you have is to use more bacterial cells for your mini prep, use a smaller volume to elute the plasmid (down to 30 ul for a mini prep) or buy a midi prep kit and chloramphenicol amplify your plasmid before running the midi. If you're using a medium or low copy plasmid, that gives a really good yield. Of course, your plasmid can't encode cat if you want to do that.
I generally add 10% 3M Sodium Acetate (pHed to 5.2, and that's 10% of the DNA volume), then 3X that total volume of 100% EtOH. I usually just throw it in the -80 anywhere from 15 mins to 2 hours (the 2 hours was because I had to run off and do something and got back late but it still turned out fine!), pellet at 4 degrees at ~16000x g for 30 mins, remove the supernatant and then wash with 70% EtOH followed by a 15 minute spin at ~16000x g. Remove the supernatant and air dry until you can't see any more residual liquid.
But as fishdoc said you can simply use more bacteria. I was using a low-copy plasmid and rather than midi-prepping and EtOH precipitating I grew up 50 mls of bacteria, and after cell lysis/neutralization etc, I pipetted it all into one column and just used an extra wash step. I know the promega column says it can hold about 15ug of DNA so you should be ok just doing that, although make sure let the column sit for a bit with whatever you decide to elute in to get the optimal amount of DNA out.
fishdoc on Jul 23 2009, 10:25 AM said:
Ms new scientist on Jul 22 2009, 09:18 PM said:
I am wanting to perform ethanol precipitation on a solution of plasmid which I had extracted using the qiagen spin mini prep in order to try and increase the plasmid concentration. I was researching on the internet and there are many protocols out there for ethanol precipiration but I was wondering what protocols most people use??? ie the most efficient.
Thank you so much for helping me out.
Htet
Add 10% volume of 3M sodium acetate, then 1.5, 2.0, or 2.5 volumes of 100% EtOH. Mix well, but gently. I've always incubated at -20C for an hour, but some I know have done it at room temp and swear it works just as well. Pellet the DNA, wash with 70% EtOH, allow pellet to air dry (but don't overdry), and resuspend in TE, tris, water, whatever. I've never done this for plasmid preps, but it works well for genomic DNA.
Another option you have is to use more bacterial cells for your mini prep, use a smaller volume to elute the plasmid (down to 30 ul for a mini prep) or buy a midi prep kit and chloramphenicol amplify your plasmid before running the midi. If you're using a medium or low copy plasmid, that gives a really good yield. Of course, your plasmid can't encode cat if you want to do that.
will there be more DNA precipitated if there are more absolute EtOH?
Allan Yue on Jul 23 2009, 12:27 AM said:
fishdoc on Jul 23 2009, 10:25 AM said:
Ms new scientist on Jul 22 2009, 09:18 PM said:
I am wanting to perform ethanol precipitation on a solution of plasmid which I had extracted using the qiagen spin mini prep in order to try and increase the plasmid concentration. I was researching on the internet and there are many protocols out there for ethanol precipiration but I was wondering what protocols most people use??? ie the most efficient.
Thank you so much for helping me out.
Htet
Add 10% volume of 3M sodium acetate, then 1.5, 2.0, or 2.5 volumes of 100% EtOH. Mix well, but gently. I've always incubated at -20C for an hour, but some I know have done it at room temp and swear it works just as well. Pellet the DNA, wash with 70% EtOH, allow pellet to air dry (but don't overdry), and resuspend in TE, tris, water, whatever. I've never done this for plasmid preps, but it works well for genomic DNA.
Another option you have is to use more bacterial cells for your mini prep, use a smaller volume to elute the plasmid (down to 30 ul for a mini prep) or buy a midi prep kit and chloramphenicol amplify your plasmid before running the midi. If you're using a medium or low copy plasmid, that gives a really good yield. Of course, your plasmid can't encode cat if you want to do that.
will there be more DNA precipitated if there are more absolute EtOH?
I have no idea about that. Usually 0.1 volume of 3M NaOAc and 2.5 volume of Abs EtOH give you good recovery of DNA
Allan Yue on Jul 23 2009, 12:27 AM said:
I have noticed no observable improvement should if one were to add more than 3x volume of 100% ethanol. So past 3x 100% ethanol, you just end up wasting ethanol.