Another possibility is that the water you are using is too pure. Several years ago people were having trouble with electroporation and someone found that making media with 99% DI water / 1% tap water solved the problem. 
Good luck,

Richard Gomer


________________________________________
From: DICTY [[log in to unmask]] on behalf of Charest, Pascale G - (pcharest) [[log in to unmask]]
Sent: Wednesday, April 16, 2014 4:45 PM
To: [log in to unmask]
Subject: [DICTY] Slow Arizonian Dicty

Dear all,

Since I moved to Arizona, our chemotaxing wild-type AX3 cells have been migrating considerably slower than they did in California, ~5-6um/min versus 10-12 um/min (the exact same cells I was using in Rick's lab). So, pretty much half the speed I expect them to move. I considered every possibility I could think of and consulted with several Dicty colleagues already, but we can't figure out what's going on. Here's what we've considered and tested so far:

- The cells: We compared the AX3 cells I moved from Rick's lab, another AX3 aliquot from Rick's lab, AX3 cells we got from the Dicty base (I don't remember the depositor fo those), and also AX3 cells I got from Chris Janetopoulos which are the same ones as the Devreotes group. They all move slower than expected in our hands.

- Speed calculations: everything seems good there, we triple checked, and made sure that it wasn't a problem at the level of the imaging software so we also measured the distances travelled using a slide with a ruler.

- The substrate and gradient: we use exactly the same dishes and coverslips, cAMP solution and micropipette to deliver the cAMP and create the exponential gradient as I used in Rick's lab.

- The temperature: Our rooms are kept to 20-22 degrees C, including the microscope room. We do observe that between room temperatures of 20 and 23 degrees, for example, the cells will move a bit faster at 23 degrees, but only to ~7 um/min instead of 5.

Of course as long as our wild-type reference and mutants are handled in the same conditions, I have confidence in our results and that we can make some conclusions, but it's driving me crazy that we can't put our finger on why our Dicty cells move slower than expected!

If you have any ideas, please let me know!

Cheers,

Pascale

Pascale G Charest, PhD
Assistant Professor
Department of Chemistry and Biochemistry
University of Arizona
1041 E. Lowell Street
Bio Sciences West, Room 345
Tucson, AZ 85721-0088
Tel Office: 520-626-2916
Tel Lab: 520-621-9926