RNase treatment - (Jul/25/2005 )

Hi
Can we give RNase treatment to PCR products before restriction digestion and ligation or it is neccessary to do gel elusion before restriction and ligation, though we are getting a sharp band on 1% gel after RT-PCR but with little amount of RNA towards the bottom of the gel.
Thanks.

hi
for restriction digestion i do not purify before. In some case of hard digestion, i precipitate (Salts Ethanol) in order to resuspend in the appropriate restriction buffer. After the restriction, i purify on gel.
That is ok for me.

Ok, as far as the RNase treatment is concerned. I add RNase A and RNase One when preparing a Midi prep. What is the difference between the two?? Will treating with only one RNase ruin my whole experiment or will it just give a partial digestion??

thanks

I don't think it's necessary to perform an RNAse treatment after PCR, as it is more likely than not, that there is no RNA after PCR amplification.

you do however need to gel extract the band to separate your product of interest from primer dimer that will also affect your ligation reaction.

Nick