It's a curiosity question. When I'm doing minipreps after pelleting the bacteria sometimes it's very easy to resuspend them in P1 (Qiagen kit), but sometimes they form a rubbery clump that is very hard to break up with a pipette and I have to vortex the tubes. It interests me because it adds more time to my lab work and I wish I could avoid it.
$\begingroup$
$\endgroup$
3
-
3$\begingroup$ how old/dead are the bacteria? resuspending fresh pellets seems easier to me..? $\endgroup$– shigetaSep 5, 2012 at 16:04
-
1$\begingroup$ Straight from 16h culture. $\endgroup$– Maciej TrybiłoSep 5, 2012 at 16:16
-
1$\begingroup$ When I do giga preps, I have to weigh the pellets and put 7g of cells into a 1L pyrex bottle, then add the P1 and a small stir bar that helps beat the pellet apart with vigorous mixing. Using more than 7g can overload the column with DNA and give a poor yield. $\endgroup$– user137Aug 30, 2014 at 7:13
Add a comment
|
4 Answers
$\begingroup$
$\endgroup$
8
This is particularly an issue when you do maxipreps and the pellet is 200x the size of the pellet from a miniprep. The reasons why? Maybe you're pelleting at too high of a g-force (pure speculation). Alternatively, you could be growing the cells too long. I typically miniprep after a 12 hr culture. If you're seeing any darker colors in your pellet, you have a significant amount of dead cells. Those dead lysed cells will spill out their genomic DNA which is very viscous.
For maxipreps, you can't break up the pellet merely by pipetting up and down a few times and calling it a day. What you can do is swirl the side where the pellet is and breakup the clumps. Alternatively you can use a different pipet tip (for an extensive discussion on this very topic see Pipetting damage to cells). I change to a P200 for my minipreps and pipetting only the area around the pellet seems to do the trick for me. For maxipreps I use a P1000 which is better than using a 5mL syringe.
-
$\begingroup$ I don't think that the pellets are darker, but I've noticed that sometimes the shaker causes my tubes to screw themselves. Could it be because they don't get enough air? $\endgroup$ Sep 6, 2012 at 11:34
-
-
$\begingroup$ I'd have to measure next time. What would you consider excessive? $\endgroup$ Sep 6, 2012 at 18:58
-
$\begingroup$ It depends on your cells and what media you're using. The OD is more to check about the aeration. A lower OD would indicate that. Too high? Maybe use more P1. $\endgroup$ Sep 7, 2012 at 6:06
-
$\begingroup$ @MaciejTrybiło more areation is better. If your tubes get lose maybe they get more shaking, therefore more areation and so faster growth. And the ratio surface for volume matters here. That's why I always use 50mL Falcon tubes even for 5mL cultures. $\endgroup$ Sep 8, 2012 at 13:20
$\begingroup$
$\endgroup$
1
What I do to avoid this is to ressuspend the cells by pipetting up and down the P1 (ressuspension) buffer on the side of the tube. This way, every time takes only a bit of cells every time and big clumps are avoided. The only times I used to get big clumps hard to ressuspend were when I went poking at the pellets first.
-
$\begingroup$ Yes, I do that too. In the situations I'm talking about, rather than slowly breaking up into a cloud, the pellet will unstick as a whole from the bottom of the tube and clog the tip. $\endgroup$ Sep 6, 2012 at 11:33
$\begingroup$
$\endgroup$
3
We routinely use E. coli preps from 16-18h overnight cultures. The cultures don't show any overt signs of lysis (sometimes you can see cellular debris if the culture has indeed overgrown). I have also noticed inconsistent times when resuspending the pellet. For my own use, centrifuging at too great a g-force or for too long can increase the likelihood of making a difficult pellet. For example, I will typically spin a miniprep culture aliquot on a refrigerated table top centrifuge for 3 min @ 5,000 g.
I always vortex my bacterial pellet to resuspend and I have had great success with this method, though I'm well aware others will pipette up and down. What I normally do to increase the ease of resuspension is to add the buffer, and let the tubes sit in the fridge for 5-10 minutes. For whatever reason, letting the pellet literally chill makes resuspension very easy relative to immediate resuspension.
EDIT: I have also observed strain-specific differences. JM109 seem a little stickier than DH5a and TOP10.
-
$\begingroup$ An interesting addendum, I can pellet my cells for 3 minutes @ 1,000 g. $\endgroup$ Oct 31, 2012 at 21:33
-
$\begingroup$ @bobthejoe - is that so? I'll have to try it next time. I've always done it at exceedingly high g forces. $\endgroup$– user560Oct 31, 2012 at 23:03
-
1$\begingroup$ since we're going into specifics with experimental protocols, I might as well get a precise answer. The Qiagen manual suggests 6,000xg for 15 minutes for Maxipreps and 6,800xg for 3 minutes for minipreps. Sounds like the geometry of rotors may make a small difference but that shouldn't affect the sedimentation velocity. $\endgroup$ Nov 1, 2012 at 19:40
$\begingroup$
$\endgroup$
Some things to consider. If you spinning too fast and too long, that is going to pack the pellet more. You can spin longer at a slower speed and you will notice your pellet is not as tightly packed. I always do 2000 rpm, 20 minutes, 4C if I'm doing a midi or maxi sized prep and harvesting bacteria in a 50ml conical.
If I'm doing a miniprep I will take the 1.5 micro centrifuge tube (with pellet and buffer p1) and scrape the bottom of the tube 1 or 2 times across all the holes in the rack. This vibrates the tube good loosening and breaking up the pellet. You can consistently perform this step across many samples, this does a good job of re suspending the bugs.
I have noticed that cultures that are overgrown, contaminated, or grown in incorrectly formulated lysogeny broth, will give the pellet some weird characteristic including color and the pellet 'texture.'
