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 <patr...@douglas.co.uk> 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 <ccp4...@hotmail.com> 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 >> > > > > -- > patr...@douglas.co.uk Douglas Instruments Ltd. > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > Directors: Peter Baldock, Patrick Shaw Stewart > > 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 > > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart 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
