Biocurious is a weblog about biology, quantified.

Invert vigorously, handle gently

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?

  1. Bill Hooker    4406 days ago    #

    Ultracentrifuge? That could be the problem; biologists typically mean spins of >100,000 x g when they say “ultracentrifuge”. You only need a few thousand x g (say, 5000 rpm with a 10cm-ish rotor) to pellet bacteria.

    You could also do your cultures the other way—grow the small prep overnight and the large prep during the day. That way the cells are log phase when you collect them. A 1:100-200 dilution of an overnight culture into fresh medium should reach log phase (A650 of ~0.6) in 4-6 hours.

    When you resuspend the cells in P1, be gentle but thorough: pipette up and down until all you get a homogeneous suspension. Then add P2 and invert the (capped!) tube, rolling it over in your hands at the same time, ~20 times. The old advice used to be “make sure the entire inside of the container is coated with slime”. Be gentle, but make sure you end up with a mostly-clear solution (it will be snotty, with some white bits, but should be mostly clear). Don’t take longer than about five minutes. Mix the P3 in the same way.

    If you’re using the cartridges to remove the SDS precipitate, try spinning instead—leave behind some of the clarified lysate rather than carry over any of the pellet.

    I suspect, though, that you’re simply overloading the column. 500 ml is a lot of bugs! Try doing a midiprep—or just using the maxiprep column anyway—with 100 or 200ml instead.

  2. kstrna    4406 days ago    #

    We typically use the Hi-Speed Maxi Kits. They work very well. When it comes to adding P1, I mix it fairly well. With the P2, I shake (inverting the tube quickly) the cell mix fairly well 6 times letting it sit for 5 minutes. Like Bill, a snotty clearish solution with barely any bits. Then I add the P3, inverting the same way & let it sit for 10 minutes. The rest follows the protocol.

    Have you tried using less culture? As Bill pointed out 500 mL is a lot. The suggested for a low copy plasmid is 250 mL. For a high copy it is 150 mL for Maxis.

  3. Uncle Al    4405 days ago    #

    Major molecular biology cohorts must be charged with creating nice colors and aromas in cultures. Gene-gineer microbial hosts to secrete anthocyanines or carotenoids for color; ionone, farnesaldehyde, or santalol for scent. It is the natural placement of tenure fast-track women and minority diversity, and where most grant funding must be directed.

  4. commenter    4405 days ago    #

    Did you put the RNAse into P1?

  5. PhilipJ    4405 days ago    #

    Thanks for the suggestions all,

    commenter—yes, there is RNAse in the P1;

    Bill and kstrna—The plasmid is rather long (14 kb), and is of reasonably low copy, so I have been trying to start out with as many cells as possible. I have contacted Qiagen and for the regular maxi prep kits they didn’t think 500 mL was too much, but also suggested I go to a lower volume and see if that fixes things. As it is low copy, I don’t suspect I’m overloading the columns, but stranger things have happened before, so I’ll give that a try.

  6. kstrna    4405 days ago    #

    It isn’t so much overloading the column just with more cell pellet you maybe putting more effort into getting all the cells lysed. In doing so, you are probably shearing some of the genomic DNA which is why it is coming down. With fewer cells, you should be able to be less vigorous resuspending and lysing your cells which will hopefully prevent the shearing.

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