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® goes with you. http://windowslive.com/Tutorial/Hotmail/Mobile?ocid=TXT_TAGLM_WL_HM_Tutorial_Mobile1_052009
