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 -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en <http://groups-beta.google.com/group/oryx_group?hl=en> [email protected] <mailto:[email protected]> Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ <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 From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of HanJie_HCT Tai Sent: 29 May 2009 21:04 To: [email protected] 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) ________________________________ Hotmail(r) goes with you. Get it on your BlackBerry or iPhone. <http://windowslive.com/Tutorial/Hotmail/Mobile?ocid=TXT_TAGLM_WL_HM_Tut orial_Mobile1_052009>
