Patrick,
This thing all things devours:
Birds, beasts, trees, flowers;
Gnaws iron, bites steel;
Grinds hard stones to meal;
Slays king, ruins town
And beats high mountains down.
Answer: TIME
When scaling up in seeding the most important factor is: "TIME"
While the environment may be the same, the rate at which it changes
is not. Since crystal growth takes TIME, the change in volume will
affect supersaturation in a time-dependent manner.
Using salt or any other parameter to compensate for TIME will
introduce an extra variable which you may not know what its effect
will be.
Sorry to mess up your solution.
Enrico.
On Mon, 19 Mar 2012 23:39:00 +0100, Patrick Shaw Stewart
<[email protected]> wrote:
Sorry, I said that last part wrong. I meant it is sometimes helpful
to*increase
* the salt by 50% when scaling up.
On 19 March 2012 22:29, Patrick Shaw Stewart <[email protected]>
wrote:
Rajesh
If you set up the volumes you suggest you will probably get
precipitation. This is counterintuitive until you realize that (as Ed
says) you will be losing a lot of protein with those small drops. When
you
scale up the surface area to volume ratio is lower, so a smaller
proportion
of the protein is lost. Therefore you go *up* on the phase diagram and
get precipitant or very small crystals.
Normally halving the amount of protein for the hits from 200 nl drops
works (suggesting that half the protein is lost from such small drops).
Try say 500+1000+500 (don't reduce the volume of seed stock because the
solution that you suspended the crystals in may be important). Or
dilute
the protein and use 1000+1000+500.
For the hits from the 450 nl drops you could reduce or dilute the
protein
by say 25.%.
Or make plenty of seed-stock and try seeding into a random screen again
with larger drops, say 1.5+1+0.5 ul
Those tiny crystals should be good for seeding, don't worry about that
(provided they are protein of course).
Streak seeding may work but bear in mind that roughly a third of the
precipitant comes from the seed stock in your 250 nl drops.
You can add the seed stock with a syringe and needle if you don't have
suitable robot ;)
Experience and data-mining suggests that reducing the salt precipitant
(in
high-salt drops) or salt additive (in PEG drops) by around 50% may be
helpful too when scaling up - I'm not sure why this works.
Good luck
Patrick
For the hits in the 250 nl drops you are probably losing
On 19 March 2012 20:31, Rajesh kumar <[email protected]> wrote:
Dear All,
I have few papers in hand which explain me about microseeding, matrix
microseeding, and cross seeding.
I have also read few earlier threads and some more literature in
google.
Using Phoenix robot, I did a matrix micro-seeding and matrix cross
seeding. I have few hits with this.
In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed)
in separate expts.
I have hard time to plan to translate this 96 sitting drop well plate
to
24 well plate to refine the conditions to get better crystals. only 1-2
hits are small crystals and they are tiny.
I wonder in 24 well plate, if I should do-
1) for Example 500+500+50nl (I am sure I cant add less that 500
nL precisely)
2) to a drop of 500+500 nL do microseeding/streaking with a hair
I appreciate if you could advise and share some practical ways to
further
my experiment.
Thanks in advance
Regards,
Rajesh
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