[ccp4bb]: 96 well xtallization trays- comprehensive reply

[Tom Brett <tbrett@pathbox.wustl.edu>]

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Here is a quite informative reply from Armando Villasenor.  It is worth
browsing through if you are considering setting up for fast
crystalization screening.  Thanks Armando!

Tom,

The message you left on the CCP4 home page was forwarded to me by Mark
Knapp, one of our team members here at Roche Bioscience.  This is what I
have found from using the Greiner and Corning crystallization plates. 
Both
are compatible with 8 channel electronic manual pipettes (see attached
text
below to see my favorite pipettes).  As far as visibility, both plates
show
good visibility under the microscope (my microscope is a wild model from
Leica--------it has better light focus and intensity than other
microscopes
in the lab).

Up to now there are no other plates besides the ones from griener and
corning-------at least that I know of.  

So far I have only used the Corning plate for setups using automated
liquid
handling.  To see more details on this, see the reference in the
attached
text below.

Good luck and let me know if you have any comments or suggestions.

Thank you very much,

    Armando Villaseñor
    Roche Bioscience
     3401 Hillview Ave.  R6E-73
     Palo Alto, CA 94304

* (650)354-7586
* armando.villasenor@roche.com R


ATTACHED TEXT:

To interested parties,

This is a summary on the materials required by the Fast Drops method and
is
intended as a supplement to Armando Villaseñor et al BioTechniques
32:184-189 (January 2002), Fast Drops:  A High-Throughput Approach for
Setting Up Protein Crystal Screens.

Fast Drops:  
Fast Drops is a method for high throughput protein crystallization.  The
method is intended to allow the setup of hundreds of crystallization
experiments in a few hours (or minutes with automation).  For example,
using
this method you can manually screen four distinct proteins against
Hampton
Screens I and II in one hour!  This includes all steps including filling
wells with mother liquor solutions as well as setting up the drops.  The
method uses a 96-well plate for sitting-drop vapor diffusion.  Several
in
house crystals that have been obtained in the micro-plate have also been
reproduced in the traditional hanging-drop 24-well plate and vice versa.

Materials Needed for Fast Drops Crystallization Setups (updated January
2002)
Novel 96-well sitting drop crystallization tray from Corning:
The corning crystallization plate is now available through Hampton
Research
(1-800-452-3899) in a 10 plate sampler pack (HR3-273) or a 50 plate case
(HR-271).
Reagent Storage Blocks plus mat and mat applicator:
Storage blocks are ideal for storing pre-made screens such as the sparse
matrix screens from Hampton Research, the Wizards from Emerald
BioStructures
etc.  Each block holds 96 crystallization conditions (2 mL each).  In
addition, mats and a mat applicator are needed to seal the
crystallization
screens when not in use. 
        Storage Block           catalog # 4222 (Matrix Tech
1-800-345-0206)
        Mats            catalog # 4422 (Matrix Tech)
        Mat applicator          catalog # 3081 (Corning Inc
1-800-492-1119)
~ $950
ClearSeal Film:
The ClearSeal Film is a transparent tape that comes already cut to the
same
dimensions as the crystallization plate.  To seal plate, you simply need
to
remove the protective film off the tape and seal.  ClearSeal film from
Hampton Research, catalog # HR4-521.
TopSeal for sealing plates temporarily (OPTIONAL):
This is a plastic with adhesive that seals crystallization plates
temporary
when filled only with mother liquor.  TopSeal removes easily, without
leaving adhesive residue behind.  This allows the researcher, if he/she
desires, to fill wells in crystallization plates with mother liquor days
before setting up protein drops.  Obtain TopSeal from Packard (cat #
6005185) or VWR (cat # CO6005185).
Electronic pipettes:
1 One 8-channel pipette for dispensing crystal screens onto
crystallization
wells (60 uL each).  Two volume ranges needed, one in the 10-200 uL
range
(for dispensing up to three plates at a time) and one in the 20-1000 uL
range (for dispensing into multiple plates).
2.      One single-channel repeat pipette to dispense protein drops
(Typical
drop = 1 each).
3.      One 8-channel pipette for adding mother liquor onto protein
drops
(add 1 uL each).  Use an 8-channel pipette with volume range 0.5-10 uL.
        My favorites (pipettes are constantly upgraded.  If you know of
better ones please let us know)
· 8-channel     20-200 µL range Eppendorf's model 4850  Brinkman
Instruments
· 8-channel     50-1200  µL range       Eppendorf's model 4850  Brinkman
Instruments
· single-channel        1-10 µL range   EDP 3 model (use LTS tips)
Rainin
· 8-channel     1-10µL range    EDP 3 model (use LTS tips)      Rainin
                Brinkman instruments: 1-800-645-3050.     
        Rainin:  1-800-472-4646.

Plexi-glass rail for Corning crystallization plate:A plexi-glass rail or
platform serves in the task of keeping track of drop location while
making
observations (see Villaseñor et al).  To obtain a plexi-glass assembly
for
your microscope, you can contact DeBolt Machine and Fabrication (408)
260-7169.

Part 2: Answers to frequently asked questions about FAST DROPS.

Question 1:
Do multi-channel hand held pipettes deliver viscous mother liquors
solutions
with accuracy?
Answer 1:
There are good pipettes and there are bad ones.  There is a great
assortment
from various vendors where later models are worlds apart in terms of
accuracy and dependability. You may have to ask your sales
representatives
to "test drive" their models to find the one that works best for you
(some
vendors such as VWR will allow "test driving").  Personally, I like the
EDP3
8-channel E8-10 from Rainin (volume range 1-10 uL).  This pipette has a
slow
mode that delivers accurate volumes of mother liquor onto protein drops
without generating air bubbles.

Question 2:
Do you get condensation on the transparent tape seal?
Answer 2:
Not anymore than tape on regular 24 well plates.  In other words,
24-well
plates will show condensation in the inner surface of the tape if left
unattended sitting directly over the light of a microscope for even a
few
minutes. (i.e. 5 minutes).  The same is true with the 96-well Corning
Crystallography plate.

Question 3:
How long does it take to observe all 96 drops on the Corning
Crystallography
Plate?
Answer 3:
When recording observations numerically (For example, using a 1-10 scale
where 1 = heavy precipitate, 6 = clear drop, and 10 = protein crystals),
you
can make and record all 96 observations in 7 minutes.  Please note: the
time
span of 7 minutes was determined from real observations using a
plexi-glass
assembly to guide the plate under the microscope (see plexi-glass
assembly
shown below).  To obtain a plexi-glass assembly for your microscope, you
can
contact DeBolt Machine and Fabrication (408) 260-7169.

Question 4:
How many proteins in your lab have been crystallized in the Corning
Crystallography plate?
Answer 4:
We have had the Corning Crystallography plate in our lab for three
months.
Since then we have obtained six protein crystals from initial screens. 
Two
of these have been further optimized for x-ray data collection.  The
other
four continue in the optimization phase.  This story of these six
proteins
captures the rational behind the 96 well plate which is to quickly
immerse a
new protein into crystallization screens where 384 conditions can be set
up
manually in one hour without physical strain.  Once a first crystal is
observed, you can continue with optimization experiments using the
traditional 24 well plate if you so desire.  
Question 5:
Do you prefer the Cybi-well over other robots?
Answer 5:
Not necessarily, we happen to have a Cyb-well robot available to us
through
a neighboring lab.  We worked with it and were surprised with the super
speed it can deliver (2800 Drops Per Hour) to our crystal screening
setups.
This robot has a small footprint and is extremely fast, quiet, and
reliable.
In addition, the robot has high precision in placing protein drops to
the
dead center of protein wells (this feature saves time when viewing drops
under microscope).   However, only one thing needs to be added to the
Cybi-well, or equivalent robots, to make us crystallographers jump with
excitement.  The Cybi-well has a tip head with room for 96 tips, which
allows for extremely fast liquid handling.  We would like a second tip
head
with room for 8 tips to do the protein handling (leaving the 96 tip head
for
mother liquor handling).  This would save on protein waste without
compromising speed a whole lot.   For more information contact 
Cybio-ag.com
OPTIONAL Comment 
Examples of robots that have both tip heads are the Cyberlab 400, the
Biomeck FK (from Beckman) and the CybiDisk (from Cybio-ag.com).  The
latter
two have a tip washing mechanism that can save on time and money.