Best way to resuspend E. coli?
Posted 25 September 2009 - 09:42 AM
I'm following a protocol that requires that I spin down and resuspend my E. coli cells a couple times. I'm working with a 1 mL culture, so I spin it down in a 1.5 mL tube. The pellet that I get is pretty darn hard to resuspend. I managed to get it to resuspend by flicking and inverting the tube. In the end, I didn't get the number of transformants that I was hoping for and I think the resuspension steps may be to blame. I have other possible suspects, but this one would be pretty easy to fix.
In my opinion, flicking looks just as bad as vortexing (which I've been told never to do in such a case). My current PI believes that working it with a pipet tip is just as bad. I personally think that I should try working it gently with as wide a pipet tip as I can find instead of flicking the bejeezus out of it.
What does everybody think? To pipet or not to pipet?
Posted 25 September 2009 - 02:27 PM
Posted 25 September 2009 - 03:42 PM
I don't want to beat up my cells because I don't want to reduce the complexity of my library. Given the size of my culture and that I'm doing this in a 1.5 mL tube, i could gently flick it all day and the pellet won't budge. I guess I could do it in a 15 mL tube.
Posted 25 September 2009 - 04:23 PM
Posted 26 September 2009 - 07:21 AM
Yeah, I can agree with this.
I have had the same problem: when spinning it down I always had the problem that the pellet was too hard and I was not able to resuspend it.
The problem is that when you have too much material you will get a pellet that is too big and too strong.
Thats why its better to work with smaller amounts and doing it a few times in stead of doing it in one time.
First of all: its better to use larger tubes: this way the pellet is spread over a larges suface, making it easier to resuspend it.
Second: fill the tube with a small amount of fluid (depends on how much material is in you fluid, so you have to fine tune this.. it can be 0.5ml, 1ml or maybe 1.5ml.)
third: after spinning it down, do not reuse this tube or remove the pellet and then reuse the tube.
If you reuse it, you will get the same problem again: pellet is too strong.
and last: maybe the problem isnt in this step, but in another step in your protocol for the transformants...
Posted 26 September 2009 - 08:54 AM
I'm going to try using a larger tube. This library creation calls for several rounds of electroporations and this is the first time I've had less than wonderful results. There are a few other parts of the transformation that could also be to blame. Our -80 freaked out last week and came up to about -60. We had to transfer everything (including the competent cells I used) to a backup
Posted 27 September 2009 - 05:19 PM
Posted 27 September 2009 - 11:56 PM
You can also spin for a shorter duration. 20 sec should be enough.