Re: [ccp4bb] about microbatch
Jiamu Although we no longer focus on microbatch-under-oil, we have a small utility called VDTOMB that converts from vapor diffusion conditions to microbatch. I have tested it with known published and in-house crystallization conditions and it seems to work remarkably well. Go to http://www.douglas.co.uk/tipsntech.htm (you must use Internet Explorer, not Firefox or something) and click on the link near the middle of the page. In most cases where the main precipitant is PEG there will be very little equilibration in the VD experiment, so the MB conditions are almost the same as the VD conditions. The typical size for manual dispensing is 1 + 1 ul. For robotic dispensing, 0.3 + 0.3 is typical. As long as you have a couple of mm of paraffin (or silicone mixed with paraffin for screening) over the drops you will be OK. If you or anyone else would like plates for microbatch, please ask me for samples - see http://www.douglas.co.uk/vb.htm. They're about $4, which is cheaper than most VD plates. MB often works well for proteins that are sensitive to oxidation, and for membrane proteins. Generally, less protein is lost on the surface of the drop, and the crystallization conditions are better known. It is also great for conditions with isopropanol or other volatile liquids - see http://www.douglas.co.uk/winner1.htm Good luck with your project Patrick -- [EMAIL PROTECTED]Douglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US 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 Jiamu Du Sent: 26 July 2008 12:39 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] about microbatch Dear all, I want to screen crystal using microbatch method. I do not have standard microbatch plate, so I have to using a 96 well cell culture plate instead. My question is below: What is a typical drop size for microbatch? 1ul protein + 1ul mother solution or larger? How much paraffin oil is sufficient for cover the drop? Thanks. -- Jiamu Du, Ph.D. State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 P. R. China Tel: +86-21-5492-1117 E-mail: [EMAIL PROTECTED]
Re: [ccp4bb] about microbatch
Hi Jiamu, I use 96-well V-well ELISA plates for microbatch and they work very well. I add paraffin until about 1mm from the top-edge of the well. You need 2-3mm of oil to reduce evaporation to very low rate. The drops will shrink over a few months as you loose water through the plastic of the plate as well. When going from vapour diffusion to microbatch it is useful to screen a range of drop ratios. I use 1+2, 1.5+1.5 and 2+1 to go from a vapour diffusion hit to microbatch. This should give you an idea of a good starting ratio for further screens. You can use any drop size you like that you can accurately pipette. Also, as pipetting error is now your main variable it is worth setting drops up in triplicate as you don't want to miss good conditions due to a bad drop. If you have crystals that grow overnight (or within a day or so) and have PEG as your precipitant then it is worth trying microbatch. Under these conditions I have often gotten larger, better diffracting crystals than trying to optimise the very rapid growth in vapour diffusiion (your mileage may vary though :) ). A problem with the ELISA plates is that it can be hard to fish crystals out of the drop using a pin. I have made some tools with 10mm steel Hampton tubes (that go into 18mm bases) and unmounted cryoloops (the ones in a paper slip) and with these you can put the tube down the side of the well to fish out the crystal (otherwise the pin base can block your view down the microscope). Some people pipette their crystals out of the microbatch experiment but I have lost a few crystals this way (the best ones of course) and so don't do that anymore. Cheers, Tom Dr Tom Caradoc-Davies Beamline Scientist Protein Crystallography Australian Synchrotron 800 Blackburn Road Clayton, Victoria 3168 Australia. Direct +61 3 8540 4187 Fax +61 3 8540 4200 Mobile +61 4 3430 7453 ** This email, including all attachments, is confidential. If you are not the intended recipient, you must not disclose, distribute, copy or use the information contained in this email or attachments. Any confidentiality or privilege is not waived or lost because this email has been sent to you in error. If you have received it in error, please let us know by reply email, delete it from your system and destroy any copies. ** From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jiamu Du Sent: Saturday, 26 July 2008 9:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] about microbatch Dear all, I want to screen crystal using microbatch method. I do not have standard microbatch plate, so I have to using a 96 well cell culture plate instead. My question is below: What is a typical drop size for microbatch? 1ul protein + 1ul mother solution or larger? How much paraffin oil is sufficient for cover the drop? Thanks. -- Jiamu Du, Ph.D. State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 P. R. China Tel: +86-21-5492-1117 E-mail: [EMAIL PROTECTED]mailto:[EMAIL PROTECTED]
[ccp4bb] about microbatch
Dear all, I want to screen crystal using microbatch method. I do not have standard microbatch plate, so I have to using a 96 well cell culture plate instead. My question is below: What is a typical drop size for microbatch? 1ul protein + 1ul mother solution or larger? How much paraffin oil is sufficient for cover the drop? Thanks. -- Jiamu Du, Ph.D. State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 320 Yue-Yang Road Shanghai 200031 P. R. China Tel: +86-21-5492-1117 E-mail: [EMAIL PROTECTED]
