Hello,
I use to perform maxiprep but recently I have had a problem while I check the DNA through a digestion in agarose gel.
I transform the plasmid in bacteria JM109, then I put a clone in a small culture in LB medium + ampicillina at 100µg/ml for about 6 hours at 30°C then I transfer into a larger culture (200 ml LB+ Ampicillina at final concentration 100 µg/ml ) for a whole night at 30°C.
The next day, I use the QIAGEN Plasmid Maxi kit. I follow carefully the protocol included in the kit. After having redissolved the DNA in water, I check its concentration that is around 1000 ng/µl ans the A260/280 are around 1.9 and the A260/230 about 2.
I use to check the DNA in agarose gel with non digested plasmid and some plasmid at around 1 µg with a mix using restriction enzymes and cutsmart buffer that I put in a hoven at 37°C for about two hours. I have added a picture with my results. I can see a band in the top of the gel that has not migrated. I noticed a smear in my non digested DNA migration. About my DNA with restriction enzymes, I can almost see a band at around 3,000 bp but there is also the big band next to the well.
Have someone ever seen that kind of result?
Thank for helping.
Cheers.
Your kit is not working here. Check the pH of your lysis buffers 1, 2, and 3. If this kit is old toss it. Your gel looks like mostly E.coli DNA.
Check the pH of all your buffers, perhaps just something got wrong.
Three decades ago I had to work in a sabotaging environment, where the profs put 3-4 people against each other - resulting in lots of potential sabotage in a many-year-K.O. project. It happened that all of a sudden my Southern blots didn't work. It turned out that my buffer for pH=7.4 was over 10 (my collaborator feared that someone may have added some DNA-denaturing NaOH-pellets). Later on, he appeared to be also sabotaged in Amsterdam where usually all K.O./ES projects never failed.
So, these things are extremely rare. I hope you find a remedy for your Maxiprep.
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