Claudia Scotti
Thu, 04 Feb 2010 05:52:46 -0800
Dear All,
Here are the main suggestions I received. I'm going to try them all.
Many many thanks for your help.
Yours,
Claudia
Possible solutions:
1. Maybe adding the cryo to the crystallization already?
2. 2R3R-Butandiol is the best cryo as being used in very low amounts. 10% are
typically enough.
3. Put a small droplet of paratone-N next to your drop such that they
touch and then drag the crystal into the oil and take care to remove the water
from around
it (the buble aorund it within the oil) and freeze. When draggin into oil go
loop first such that
the loop plows the way a bit (or some part of it) mechanical sress can be a
problem due to
the viscosity, but otherwise it often works, though not always..
4. 2M amm sulfate might be quite enough if you do the transfer and dipping
quickly.
5. other option is to add salt. e.g LiSO4 should be good cryoprotectant (see
cryosalts)
at high conc.
6. Have you tried with ethylene glycol for example?
7. Maybe you do not need any cryoprotectant and crystals mountd in small loops
will freeze just fine straight out of your drops...
8. Under what conditions did the crystals grow? If its sodium acetate and
ammonium sulphate I would try adding sodium malonate as a cryo
protectant (with 2-2.3M ammonium sulphate adding 0.6-0.8M Na malonate
should be sufficient) or increasing the ammonium sulphate concentration.
Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia
Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039
0382 986335/8/1 Facs 0039 0382 303673
From: lp...@cam.ac.uk
To: claudiasco...@hotmail.com
Subject: Re: [ccp4bb] Freezing under oil
Date: Thu, 4 Feb 2010 11:52:03 +0000
Could you try using ammonium sulfate as your cryoprotectant? What happens if
you slowly increase the concentration over time to >3M, followed by flash
freezing?
What about other cryos, like ethylene glycol?
lpj
Lauren Jackson, PhD
lp...@cam.ac.uk
On 4 Feb 2010, at 09:54, Claudia Scotti wrote:
Dear list,
I'm trying to freeze crystals in cryoconditions containing the following:
0.1 M Sodium acetate pH 4.4
2.15-2.3 M Ammonium sulphate
7% n-butanol
15% glycerol
The problem is that the crystals (beautiful hexagonal prisms) seem to shatter
in a random fashion: some are unaffected, some, coming from the same drop,
disgregate miserably. One even split into three parts, of which two disgregated
and one survived perfectly well.
I've tried both by moving the crystals directly in the cryocondition and by
progressively increasing the glycerol concentration to no avail.
Shall I just select those that survive?
I was wondering if anybody has ever had this same problem and if freezing under
oil could be an alternative. If yes: any suggestions on how to fish the
crystals? I tried it in the past, but I found it very difficult...
Experiences and suggestions are welcome.
Claudia
Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia
Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039
0382 986335/8/1 Facs 0039 0382 303673
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