7 replies to this topic

[GradMom]

Hi all!

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?

Thanks!

[bob1]

I take it you are making competent cells?  If so, the resuspension is important, you need to do it gently, and it will take some time to do so.  Gentle flicking is probably the best way to go about it, though using a wide bore pipette (e.g. pastic pasteur) should be OK.

[GradMom]

I'm actually making a randomized binding site library for a bacterial one-hybrid assay.  The part in the protocol that requires multiple resuspensions is towards the end where I've got my library in E. coli and I am plating them on selective media to remove self-activating sequences.  I start out by electroporating my library into E. coli.  Then I add 1 mL of rich media (SOC) and incubate for 1 hr to allow for recovery.  I then pellet, add minimal media (YM) with my selective agent (Kanamycin), and allow it to adapt to the new growth conditions for one hour.  I then pellet, wash with sterile H2O twice, pellet, and add minimal media (YM) without the selective agent.  I then plate on selective media (FOA + Kan).  

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.

[phage434]

Use a 2 ml tube, which makes things much much easier.  Also, add only a small amount of liquid at first, resuspend, and then add the remaining liquid.

[pito]

phage434, on Sep 26 2009, 02:23 AM, said:

Use a 2 ml tube, which makes things much much easier.  Also, add only a small amount of liquid at first, resuspend, and then add the remaining liquid.

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...

[GradMom]

Thanks everybody!  

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  

[swanny]

Spin at moderate speed (say 50% of maximum), and the pellet won't be so solid.

[perneseblue]

swanny, on Sep 27 2009, 05:19 PM, said:

Spin at moderate speed (say 50% of maximum), and the pellet won't be so solid.

You can also spin for a shorter duration. 20 sec should be enough.