wonder Posted October 7, 2004 Share Posted October 7, 2004 hi guys! how can i determine the hybridisation's temperature empirically? is there a website for doing this or any tricks? hopes for replies! thanks alot! Link to comment Share on other sites More sharing options...
daisy Posted October 8, 2004 Share Posted October 8, 2004 For in-situ hybridisation, FISH, southern, Western, Northern....what? And anyway, there are horrible calculations you can use but I never do, I just optimise....try a molecular biology protocol and discussion forum. Link to comment Share on other sites More sharing options...
wonder Posted October 9, 2004 Author Share Posted October 9, 2004 hi daisy! it is southern hybridisation. so you think we shouldn't calculate, but optimizing? how can i optimimize? thanks again! Link to comment Share on other sites More sharing options...
daisy Posted October 9, 2004 Share Posted October 9, 2004 Well you do that by trying different temps and wash stringencies...as I there are calculations you can use based on melting/annealing temperatures but I usually just try a range of temps. for hybridisation. Southerns are not really my area...I know about Northerns and in-situ hybridisation. I'm not by any means saying you shouldn't calculate...I'm just saying that I don't. If you can get access to a copy of Maniatis/Sambrooks lab manual they will no doubt have formulae that will help you achieve a range of temps to try ....I have my own set of volumes so if you are prepared to wait until I'm back at work after the weekend I can look this up and see if they have any tips on this topic. Link to comment Share on other sites More sharing options...
wonder Posted October 10, 2004 Author Share Posted October 10, 2004 thanks daisy! i will wait Link to comment Share on other sites More sharing options...
daisy Posted October 11, 2004 Share Posted October 11, 2004 Try this web page: http://www.sigmaaldrich.com.au/tec_mel.htm. Maniatis has a similar formula but you nedd to remember that if you use formamide you can decrease the hybridisation temperature so they say: The Tm is depressed by 0.63 degrees for each percentage of formamide where the G+C content of the probe and target is 30-75%. Hope that helps a bit. Link to comment Share on other sites More sharing options...
wonder Posted October 11, 2004 Author Share Posted October 11, 2004 thanks! Link to comment Share on other sites More sharing options...
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