Hi

 

I have a query about ordering DNA substrates for NMR...According to literature for a 1:1 ratio DNA:protein I need to titrate 10mM DNA substrate per NMR reaction. Most of the facilities that offer synthesis of oligos produce uM concentrations. Where can I order these quantites of DNA (in the UK), for NMR (I am also assuming that they need to be HPLC purified?)?

 

Any help would be muchly appreciated

Hi

 

I have a query about ordering DNA substrates for NMR...According to literature for a 1:1 ratio DNA:protein I need to titrate 10mM DNA substrate per NMR reaction. Most of the facilities that offer synthesis of oligos produce uM concentrations. Where can I order these quantites of DNA (in the UK), for NMR (I am also assuming that they need to be HPLC purified?)?

 

Any help would be muchly appreciated

 

I'm confused. First of all what matters is the molar ratio of DNA to protein... So just use an equal molar concentration of DNA that you're using for your protein.

 

Also usually when you order oligos they come as a lyophilized powder so you can disolve them yourself and select your concentration.

 

The type of purification you get usually depends on the length of your oligo. As there is a small error associated with every step in the synthesis these accumulate thus longer oligos will have more erros. I don't do alot of nmr so I'm usure what the tolerance of the procedure is.

However if you don't mind paying the extra bucks I say pay for PAGE purification. Or you could just do it yourself, that's what i do.

 

(Also the girls next door who do x-ray cystollography do PAGE purification of DNA so it should be good for your purposes.)

 

Typically if I start out with with a 50 - 100nmoles order (ie enough substrate to synthesis 100nm of oligios). I get ~50nmoles back after purification, redisolve that in 100uL and get a final concentration of 5mM. So you can see how getting 10mM isn't really a challange.

 

This is still typically far to concentrated for my purposes and has to be diluted far more.