Annealing plasmid DNA - (Jan/16/2008 )
Please help me! I am wondering if anyone has any advice on how to anneal plasmid DNA 6.4kb.
I was trying to perfect my gel purification techniques when I came across this article:
I followed their 6th tip: " Renature the DNA. The melting step combines high amounts of chaotropic salts with heat. This combination will denature the DNA. If the eluted DNA appears half the expected size (it is now single-stranded), re-nature the DNA by warming up to 95C for a minute and let cool slowly to room temperature. "
And now I have mostly single stranded DNA: One reaction where I used T4 DNA polymerase to blunt end my vector resulted in degraded DNA (exonuclease activity on SS DNA?). In a second experiment, I ran out a gel on my inserts that I had just gel purified. I cut out a 1.2 kb band, I then tried to "renature my DNA", and then when I ran another gel before any further enzymatic reactions I got two bands on the gel. One 1.2 kb and one 0.7 kb.
Please help me renature my DNA!
This seems quite complicated -- what is your ultimate goal? Why do you need to renature your DNA? If you are struggling, perhaps we can suggest an alternative method. I'm not familiar with the technique you're describing for a simple gel purification (renaturing).
Yes, please do explain what you are trying to do. Why are you trying to renature your DNA? Why have you exposed your DNA to reagents which requires you to renature your DNA in the first place? And what might these reagents be?
Unless you have supercoiling in your DNA, or your fragments are small, I don't think it is possible to renature DNA of such a size
(6kb) can be done accurately. I believe the best move would be to start again from your plasmid DNA.
Thank you for your posts. I agree, It was a stupid thing to do. I'm not sure why I listened to the bulletin to begin with.
I was just trying to be perfect with my gel extraction of my vector. (Which I should not have done since my gel extractions are pretty good as is). Since the bulletin I cited says that the chaotropic salts that are used to get the DNA to adsorb to the Qiagen silica column may denature the DNA, one should heat up the DNA to 95 and then slowly cool it down to room temperature. The problem was that when I checked my DNA (OD, ethidium bromide gel) much of it was single stranded.
I know that it is possible to anneal oligos, and though it may be possible to anneal the plasmids by incubating it longer at its Tm. That would not get around my greater fear, that the two strands of single stranded linearized plasmid would not reanneal with each other perfectly. There are 7000 bases in my vector. It seems more likely to me that with a molecule that long it would be easier for it to kink up on itself then to align perfectly with another strand of 7000 bases.
Anyway, I decided to cut my losses and I'm digesting fresh plasmid as we speak. This time around though, I'm not going to try and improve on what works.