3 replies to this topic

[relevant_username]

Hi,

 

I am receiving abnormally low yield when using the Qiagen midi prep to isolate DNA from an overnight culture. Typically, the yield for a mini or a midi prep is in the neighborhood of 400-500 ng/ul of DNA, or higher. However, I am currently receiving a maximum of 150 ng/ul, with concentrations as low as 10, 20 ng/ul, practically nothing. I've isolated similar plasmids before in this lab (this new construct that I'm trying to isolate is literally one point mutation different) and have gotten much better results than this, so I don't know what the issue could be. Another important fact is that the 260/280 ratio for these purified DNA samples is way too high: I'm getting readings of 1.91 and up when I normally see a range of 1.81 to 1.87 at the highest.

 

I don't know what exactly the issue is because everything seems normal...the overnight culture looks fine, I'm seeing a nice fat pellet after spinning it down. I have been following the Qiagen midi kit manual to the letter. Maybe I'm missing some subtleties that are causing my DNA to disappear, but I cannot imagine what it might be. Any advice/tips/help would be appreciated. Thanks.

[Missle]

I don't consider a 260/280 ratio of 1.91 to be too high.  I routinely get this ratio and I believe it to be normal.  Qiagen lists their 'typical' ratios as 1.7 - 1.9.  Is the copy number of the plasmid the same as your previous constructs?

[relevant_username]

Yes, copy number should be the same (low-copy). Maybe a 260/280 ratio of 1.91 is okay, but since it is different from what I'm used to seeing, I thought that it could be an indication of something significant, which might be related to my low yield issue. I'm trying to figure out if there is a step in which my DNA is vanishing, or if there is a problem with my cell culture. Is there anything you would try in my situation?

[Missle]

What OD600 is your culture getting to?  Is it possible that there is a problem with your antibiotic and your losing the plasmid during culture?  As long as I was following the instructions of the Qiagen kit and the kit isn't expired or anything, I've always found them to be reliable so I would poke around more on the culture side.  The stock that you're using to generate your culture, is it a glycerol stock?  Maybe try taking a scraping and plating it out on a fresh antibiotic plate, choose a colony and start from there.  Otherwise, try going back and do a fresh transformation into a storage cell line if you're still having problems.

 

Good luck!