Maxiprep troubles - loss of DNA at last stages of maxi prep (Mar/21/2005 )
I'm having touble with my ceasium chloride method maxi preps. All seems fine untill the very last ethanol purification stage. It seems as if I have a large DNA pellet untill I attempt to desalt in ethanol (70%), after which the pellet seems to have dissapeard.
Is it possible that something is going wrong in the butanol extraction step?
Are there seminal pionts in this protocol which I should be extra vigilent about?
Any help appreciated.
i can't figure dna dissapearing in ethoh 70. once i've used a wrong concentration and loss my prep.
dna does not dissolves itself at ethanol 70%maybe you can prepare a fresh 75%solution. 75% is ok to wash from salts.
you can see this topic too
A thing to note with 70% ethanol washes ... the DNA can become more translucent and therefore may seem to disappear but in reality it is still there.
Usually when we see a pellet, it is salt, which is diluted away in the 30% water of the 70% ethanol wash.
When does the pellet disappear - after the centrifugation or after you dry the pellet?
Have you tried quantifying or running some of the end-product on agarose to check if you actually have plasmid?
Also, do not over dry the pellet - an overdry pellet will also seem to "disappear". Make sure you allow the DNA to dissolve when you resuspend it ... leave overnight if possible (at 4C).