hybridisation's temperature empirically?

[wonder]

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!

[daisy]

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.

[wonder]

hi daisy!

 

it is southern hybridisation. so you think we shouldn't calculate, but optimizing? how can i optimimize?

 

thanks again!

[daisy]

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.

[wonder]

thanks daisy! i will wait

[daisy]

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.

[wonder]

thanks!