by PhilipJ on 28 July 2006
Plasmid purification isn’t all that hard, except that when you’re in the physics department and you’re using the shared use facilities in the molecular biology building. But anyway.
I’ve been trying to get a clean sample of my pPIU1 plasmid, and I need a fair amount so I’ve been using a QIAGEN maxi prep kit to do so. The basic idea is that you pick a single colony of cells from an agar plate and grow them up in liquid media (~ 5 mL) for about 8 hours. You then take a small volume of this liquid culture and use it to start a new, large (~500 mL) liquid culture which you leave growing overnight. By the morning you have a nice cloudy (and smelly) culture of cells that are ripe for harvesting.
You collect them by spinning the liquid down and forming a pellet of cells, which you can then resuspend, lyse, and harvest the DNA from. It involves lots of time standing around ultracentrifuges (no MRF yet, thankfully), and as long as you can follow the recipe, everything should go smoothly.
The problem I’m having, however, seems to be genomic DNA contaminating my end sample (see the gel at right, there’s a long peice of something which hangs out up in/near the well, and if I digest the sample (not shown) I get horrible streaks all along the lane). The QIAGEN Plasmid Purification Handbook (PDF) gives instructions on how to lyse the cells to begin with, (steps 5 and 6 on page 21) which tells you to “mix thoroughly by vigorously inverting” 4-6 times, with two different buffers. These are the steps which actually break the cells apart, making it possible to then separate the DNA from the rest of the cell soup. When you have problems like I’m having and go to the comments and suggestions section (page 38, under Contaminated DNA/poor-quality DNA), they say if there’s genomic DNA in the eluate, they tell me the mixing of the bacterial lysate was too vigorous! In fact, it must be handled gently.
Great. Thanks QIAGEN.
In my defense, I’ve done this a couple of times now and I’m sure I’ve payed attention to how rough and tumble a life my cell lysate has had, and I seem to end up with genomic contamination every time. It doesn’t seem to show up on mini preps of the same cell culture. I’ve also tried two different maxi prep kits to see if there was something wonky with the buffers, and that doesn’t seem to be it either. Any wet lab experts have any suggestions?