You can also try a "miniprep" of your transformed cells. I will also try precipitating it. So my questions are, what has happened to the DNA? I was kindly sent a plasmid from an overseas researcher, successfully eluted using ddH2O and transformed bacteria, getting many colonies. I tried transforming bacteria again and only got colonies for one of the constructs I have 3 constructs total.
Did you elute the DNA from the entire piece of paper? But I'm hesitant to use all of my elution.
Ampicillin is bacteriostatic, so it won't kill the bacteria, just stop them from growing. I would definitely not precipitate the DNA, since you may already have such a low concentration of viable plasmid that you will loose the rest of it trying to concentrate it.
There may be enough non-degraded copies of the plasmid left to get a successful transformation when you concentrate the DNA? Felicity -Felicity185-. I think I might add several hundred microlitres more water, mash it up some more and then aim to use half to three quarters of that volume for the precipitation step.
You need to give them time to express the resistance genes that allow them to survive your selection agent.
And yes, there is a good chance the DNA has degraded- and you can't rescue it after that. Fingers crossed!. Please help! New Forum Archives 2009-: There might still be plasmid in the cells. However I was promptly told by my supervisor to drop everything I was doing and focus on writing my thesis. Thanks for the reply leelee. Unfortunately I eluted from the entire spot of DNA.
If the later, I would elute some more and try a transformation with that. I transform by heat shocking the bacteria... Molecular Cloning. As a last resort, you can design primers and clone the insert into new vector.