midi-prep - (Oct/24/2013 )
I did the midiprep. I precipitated the DNA with isoproponol and I can see some precipitate at the top, somehow when I try to tranfer it to the other tube for the centifugation, it disappear and after centrifugation, i did not see any pallet???? and I contniue, I try to dissolve everything in the ethanol, as I hope there is somethnig, may be i cant see it and I centifuge it again and dissolve everything in the water, I run the gel and i dont see any DNA there, Where went the DNA???
Add a few microlitres of NaCl and see if there is any precipitation.
how much concentration of NaCl. and add the NaCl in dry tube, suppose to have DNA pellet???
After running on gel if you have saved any amounts in the tube, to it add NaCl (0.2M)
I run the gel, I can see some very little amount of DNA...I measured the concentration and its 20 ng/ul. Its even low then miniprep....I dont know whats wrong happened with the plasmid....
Is this a kit based extraction or a crude phenol-chloroform extraction?
When DNA concentrations are low, you can precipitate your DNA out with 10% NaOAc/EtOH for 30min to O/N at -80C. You also add glycogen if your ethanol solution if your DNA is being difficult.
Edit: Oh the typos...
Were you able to do the midiprep of this plasmid successfully in the past or is it the first time that you are working with this plasmid?
Did you have any problem to grow the culture? Which antibiotic are you using? Is it ampicillin? If it's ampicilin, try changing to carbenicilin.
I once had problems with a plasmid at the time of doing maxipreps: the culture growed very well, but after the preparation there was no DNA or a very very little and useless amount.
My plasmid contained a gene for ampicilin resistance, so I solved the problem just by using carbenicilin instead of ampicilin. On some occasions, when plasmids are "toxic" for the baceria, bacteria express ß-lactamase that degrades ampicilin in the culture and this way they can get rid of the plasmid, because they don't need it any more, and start growing without it. This way, when you do your midi/maxiprep you get hardly no DNA, if any. Well, more or less I think that's the way it works. Carbenicilin is less easily degraded an therefore bacteria should conserve the plasmid in order to grow.
Just an idea.
the kis is based on Isoproponal precipitation, once I added the Isoproponal I can see the precipitates but they just disappear in few minutes and if I centrifuge it I dont see any pellet. Its first time I am doing the midiprep for this plasmid. Its just pET16 vector with out any gene insert into it. I just wanted to have a plasmid for my later stuff. Culture grows very well.
Pellet disappearing might be only salts that disolve... not DNA
So where is DNA then???