Re: [ccp4bb] Seeding

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Postdoctoral Positions in Ion Channel Structure at UCSF
From: Dan Minor daniel {- dot -} minor {- at -} UCSF {- dot -} EDU
Date: 2009-06-03

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From: Martyn Winn martyn {- dot -} winn {- at -} STFC {- dot -} AC {- dot -} UK
Date: 2009-06-03

Subject: Re: Seeding
From: Patrick Shaw Stewart Patrick {- at -} DOUGLAS {- dot -} CO {- dot -} UK
Date: 2009-06-03

HanJie

The easy way to seed either by hand or using a robot is to use the
approach of D'Arcy (ref below)

Comments:

1. Seeding into SCREENS will give you new leads and may solve your
twinning problem

2. If you are seeding into screens DON'T DILUTE THE SEED STOCK.
The more seeds you put in, the more new hits you're likely to get. Only
use dilutions if you get too many crystals

3. You can make the seed stock using the reservoir solution that
gave the hit - you don't need to have protein present or to increase the
precipitant. However the seeds are often unstable so you must KEEP THEM
ON ICE all the time and freeze them as soon as possible. They'll then
last at least a year.

4. You don't need to fish out crystals. You should put the whole
contents of the well into the seed bead tube, including precipitant,
crystals, dust etc

This method has given outstanding results and is very popular

Patrick

[2] Allan D'Arcy, Frederic Villarda, May Marsh. 'An automated microseed
matrix-screening method for protein crystallization'. Acta
Crystallographica section D63 (2007), 550-554. On-line at
http://scripts.iucr.org/cgi-bin/paper?S0907444907007652

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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
HanJie_HCT Tai
Sent: 29 May 2009 21:04
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Seeding

Hi,

I set up some seeding trials (microseeding) from 1 x to 1000x dilution.

I am just wondering

(a) How long the seed stock solutions can be kept? some said they can be
kept for up to 1 year, some said the seed stock solutions need to be
freshly prepared before use, which one is true?

(b) Each well I set up two drops, and then I streaked these two drops
continously. Between each well (means each dilution microseeding) do I
need to use ethanol or water to rinse the whisker? Or I can just
continue to streak seed as long as the sequence is from low dilution
(1000x) to high dilution (1x).

(c)From my result, how come some crystals showed up in 10 X dilution
while some showed up in 100X and even 1X while other no crystals were
grown. The result wasn't consistent. On the same well, sometime 1st
seeding drop got crystal, and someting 2nd seeding drop got crystal, Is
this phenomenon common?

(d)After trying 1-2 rounds of microseeding, the crystal remained as
twinned or multiple plates overlapping with each other? Any suggestion
to improve the twinning crystal condition?

My microseeding protocol is as followed.
(1) loop up a crystal either from normal optimization or seeding drop
(eg 23% precipitant), size may be 0.1 mm.
(2) transfer to slightly 5ul dissolving solution (eg 17%
precipitant)...30seconds
(3) transfer to 50 ul stablizing solution (normally I used the same
solution from equilibrium well, eg 23%)
(4) use pipet to withdraw all 50ul solution that contains the crystal
(5) transfer to 1.5 ml seed bead centrifuge tube (Hampton Research)
(6) vortex 90s
(7) add 450 ul same stablizing solution; this is 500ul seed stock (1:1).
Sometime, I manipulated the final volume a little bit, such as 100ul or
300 ul.
(8) transfer 5 ul 1:1 seed stock to another 45ul stablizing solution
(9) vortex 90s
(10) The final 50 ul seed solution became 1:10 dilution stock.

Regards,
HengChiat Tai (HanJie)

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CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Postdoctoral Positions in Ion Channel Structure at UCSF
From: Dan Minor daniel {- dot -} minor {- at -} UCSF {- dot -} EDU
Date: 2009-06-03

Next message:
Subject: 3 positions available in the CCP4 core group
From: Martyn Winn martyn {- dot -} winn {- at -} STFC {- dot -} AC {- dot -} UK
Date: 2009-06-03