need help to solve problems with plasmid isolation - molecular biology (Nov/20/2007 )
Hi to you all,
I tried to perform plasmid isolation by using kit produced by nucleospin.
Although I tried to carry out plasmid isolation at several times, in all cases I couldn't obtain enough concentrated plasmid DNA. The amounts were close to 80 nanogram/mikroliter.
Plasmids contain ampicilin gene in them, and I prepared LB medium with ampicilin. I applied appropriate time for growth of bacteria.
Thus the problem isn't related with low amount of cells or cells without plasmid. Morover plasmid origin of replication allows high copy number of plasmids.
I applied procedure without making any change, I even performed optional steps.
I changed elution buffer with dd H2O in order to eliminate possibility of out of order elution buffer. (in protocol it is stated that dd H2O can be used instead of elution buffer) But nothing changed.
What are the possible reasons of not having plasmids?
If you have any suggestions I will be glad of hearing them...
Qiagen kit is what we use. Never failed once on my hand.
If you have small samples saved each step and assayed for the presence of the plasmid, a diagnosis is possible. Otherwise, too many things can go wrong.
what is the nature of your plasmid is it high copy number or low copy number? what is the replicon ? read molecular cloning by sambrook et al. It is a very good protocol book we are using the protocol of alkaline lysis mentioned in this book and usaly gel 5-7 microgram of DNA per ml of original culture.
80 ng/ul is a fine concentration of plasmid. Why do you think you need higher concentrations? I would stick with EB for elution, or work with TE rather than water. The pH of water is poorly controlled.
I agree that 80 ng/ul is a good yield. However, if you think that you really ought to be getting more, you might try warming up the elution buffer slightly. I do this with the Qiagen kit and it usually helps increase my yield.
I agree with others that 80ng/ul is decent for miniprep DNA.
You might want to heat the elution buffer or TE upto 60C and use it for elution. Might increase yields.
Are we talking about mini- or maxiprep? For Maxiprep the DNA-amount is not optimal. We use Nucleospin kits as well and they work fine for the most plasmids, but I have a very stubborn one which gives not so good yields. You could try to grow your bacteria in TB instead of LB and at 30°C, this helps sometimes. A second try could be to do the elution one time with half of the elution buffer volume, then a second time with the other half.