maxiprep help - (Sep/20/2005 )
I used Qiagen maxiprep kit to prepare plasmid. I can not figure out anything wrong in the process, but the 260/280 ratio is only 1.45, although the concentration is 1.3ug/ul in 400ul water. What cause the 260/280 ratio so low? What can I do to further purify the DNA?
first i would heat 50° 10' the plasmid after elution in order to accurate the OD.
For further purification, proteinase K treatment, then phenol chlo followed by alcohol precipitation will do the job.
You most likely overloaded your column with too much bacterial lysate or had contamination from the precipitate carried over to the column, thus have protein contamination. You can try a phenol/chloroform extraction to further purify this DNA, otherwise try to reduce the amount of bacteria and be careful wyou use for the prep in the future.
I tried 3 times, and using less bacterial lysate, but still got the similar 260/280 ratio which is about 1.4-1.5. The second time, I repurified the DNA with a new column according to Qiagen protocal, but still got the same result. The 3rd time, I further purified the DNA with phenol/chloroform protocal, but the 260/280 ratio was still 1.4-1.5. I never had such problems before.
are you sure it's bad? have you run a gel? perhaps your bulbs are burning out, or some other problem with the spec?
Your DNA should be fine if you repurify it. I would check your spec to see if their is an problem.
Insanity: doing the same thing over and over again and expecting different results. -- Albert Einstein.
What do you intend to use the DNA for? I know a lot of you will find this shocking, but I haven't done an A260/A280 ratio in like fifteen years... I find if I have enough DNA by inspection of a gel, things work fine. Mind you, DNA is my bread and butter -- I'm a molecular biologist working on genetics of bacteria -- it seems all I do is clone stuff, PCR stuff, move DNA around, etc.
In like fashion, I've never calculated inset to vector ratios, picomoles of ends, and other such calculations good people get hung up on every day. I have not (nor anyone else in my lab) ever found any of this stuff to be necessary, and we've been pretty successful...
I agree with you, homebrew
I do mostly protein work, emsa, and qPCR these days, but i do a little cloning (and I used to do a LOT of cloning) and I never have spec'd my DNA
the only thing I bust the spec out for is to check my RNA preps before moving on to RT
BTW, I hope my prior post does not sound insulting to anyone -- that was most assuredly not my intention.
Homebrew, I've got to disagree with you on this one bud. I think the purity of the DNA does make a huge difference if you are doing things like transfection, PCR, ect. Running it out on a gel just frankly just wastes time. Things like not checking on the vector to insert ratio I can see if you have lots of experience. It is like being able to cook without a cookbook because you know how to adjust the recipe. For the most part when I have students who do not care to spec. the DNA or RNA, I get pissed because when it comes to nucleic acids I always think in quatitative terms. Just my $0.02