hybridisation's temperature empirically?

[indoubt]

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!

[Monique]

Do you mean DNA melting temperature? There's a simplified formula you could use for an estimate, but this works better http://scitools.idtdna.com/Analyzer/

[iansmith]

What type of hybridization are you using?

[Moonbear]

When you say empirically, generally that means trial and error. You can use a method to calculate a likely starting point, then use your results to adjust the temperature up or down to adjust your stringency. If you have a lot of background along with signal, increase stringency, if you aren't getting any signal, decrease stringency.

I didn't look at the site Monique provided, but if it calculates the melting temperature, you can try starting off with a hybridization temperature about 30 C below the Tm. Your hot wash temperature will also affect stringency, so you need to adjust both hybridization and hot wash temperatures appropriately (this makes it doubly challenging).

[indoubt]

What type of hybridization are you using?


hi Ian!

it is a hybridization between two DNA molecules. we are looking for if they have any homology.

[indoubt]

When you say empirically, generally that means trial and error. You can use a method to calculate a likely starting point, then use your results to adjust the temperature up or down to adjust your stringency. If you have a lot of background along with signal, increase stringency, if you aren't getting any signal, decrease stringency.

I didn't look at the site Monique provided, but if it calculates the melting temperature, you can try starting off with a hybridization temperature about 30 C below the Tm. Your hot wash temperature will also affect stringency, so you need to adjust both hybridization and hot wash temperatures appropriately (this makes it doubly challenging).


hi moonbear!

you said there is "a method to calculate a likely starting point" do you know which method? it would help alot, i think.


by the way is hybridization's temperature the same as annealling temperature in PCR? it seems like that, since in PCR the primer will anneal (hybridize) to the template molecule.

so does it mean that we don't have any calculate machine for hybridization's temperature?


thanks!

[Moonbear]

indoubt, when I read your post earlier today, I thought you were talking about in situ hybridization (I must have just added those words in my own head as I read). I don't know the answer for your application. It may be the same, but I'm not sure. Does Monique's link include a calculator? I haven't checked it yet.

[indoubt]

hi moonbear!

it is in situ hybridization and not PCR i was talked about. i mentioned about PCR bacause i just wanted to compare the annealing temp. in PCR and hybridization temp. in in situ hybridization.

hope i don't confuse you!

[iansmith]

You might want to look at this
http://www.roche-applied-science.com/fst/products.htm?/DIG/dig_hints04b.htm

[indoubt]

thanks! :smile: