Hello Scott
Setting up rMMS by hand works fine, but it's a bit slow and uses more protein and (sometimes much more important!) more seed stock.
We recommend using a Hamilton syringe, preferably with a rounded needle, to set up by hand. 1.0 protein + 0.7 reservoir solution + 0.3 seed stock works well. Dispense the protein and reservoir solution first, then add the seed stock You can clean the needle by passing it through the reservoir just before you dispense seed stock. It's surprising how accurately you can dispense the seed like that with a syringe and your fingers.
I can't say whether dispensing by robot or by hand gives better results. The most productive group that I know that used this method had great success by hand before started using a robot, so I'm sure that both work well.
Bear in mind that you're likely to get showers of small crystals at first when you use this method. So you will probably need to dilute the seed stock. What works extremely well for this is our new "Combinatorial" script. Here you arrange the hits in columns, and add a different seed stock to each row. Make a dilution series from "neat" seed stock to say a 1:100,000 dilution. Add the most dilute seed stock to the first row, the next most dilute to the second and so on. Its a really quick way to get 1-5 crystals per well, and you can easily set up a whole plate with almost the same number of crystals per well. We use the robot to do this but I'm sure it would be easy to do the same thing by hand.
Best wishes
Patrick
On 24 May 2013 13:46, Scott wrote:
Dear Patrick,
Have you had better optimization using MMS by setting up trays with a robot or into
larger sitting drops by hand?
Thank you for your suggestions.
Cheers,
Scott
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wrote:
Use "random" microseeding to pick up new conditions, and work with those.
On 24 May 2013 03:20, Urmi Dhagat
<[log in to unmask]> wrote:
Hi,
I am working on a protein antibody complex which readily crystallizes (crystals form overnight and grow over 2-3 days) in 0.1M Bicine pH 9, 10 % PEG8000. The crystals are chunky - shaped like a parallelogram but they diffract poorly to about 8 Å.
I have tried the following to improve diffraction:
1. Screen different temperatures 4°C - crystals have bad form and 10°C crystals grow slower but diffraction does not improve.
2. I have done an additive screen – A few hits came up like Yttrium Chloride and Acetonitrile but they don’t improve diffraction either
3. I have tried streak seeding this does not help either
4. Tested different cryo protectants – MPD, PEG400, Ethylene glycol and glycerol - 10 - 15% glycerol seems to work best
5. Not sure if cryo protectant affects diffraction in this case – I will look at room temp diffraction soon to rule this out.
6. Typical diffraction images attached
Does anyone have suggestions on what I could try to improve diffraction of my crystals?
Urmi Dhagat
St Vincent's Institute
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[log in to unmask] Douglas Instruments Ltd.
Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
Directors: Peter Baldock, Patrick Shaw Stewart
http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034
Regd. England 2177994, VAT Reg. GB 480 7371 36
