Protocol for DNase I treatment of RNA - (Nov/22/2005 )

Hi,

I am a non-molecular student so would appreciate help with this!

I need to treat my RNA extractions with DNase I (Roche) but can't find any instructions (with the product or online) on how exactly I do this.

Can I just add the DNaseI straight into my RNA extract, and if so how many units? Obviously I also need to work out how much RNA is in my sample, but I will have a small sample size (100uL) and don't want to sacrifice a lot of it. Should I just dilute it say 1 in a 1000 and use a spectro?

Any suggestions greatly appreciated.


Cheers

Hi boo,

This is a protocol adapted from invitrogen (http://www.invitrogen.com/content/sfs/manuals/18068015.pdf)

DNase I treatment of RNA
Add the following to an RNase-free, 0.5-ml microcentrifuge tube on ice:
1 µg RNA sample
1 µl 10X DNase I Reaction Buffer
1 µl DNase I, Amp Grade, 1 U/µl
DEPC-treated water to 10 µl

NOTE: To work with larger amounts of RNA, scale up the reaction (including
volume) linearly.

Incubate tube(s) for 15 min at room temperature.
Inactivate the DNase I by the addition of 1 µl of 25 mM EDTA solution to the reaction mixture. Heat for 10 min at 65°C.

NOTE: It is important not to exceed the 15-minute incubation time or the room temperature incubation. Higher temperatures and longer time could lead to
Mg++-dependent hydrolysis of the RNA. Additionally, it is vital that the EDTA be added to at least 2 mM prior to heat-inactivation to avoid this problem.

10X DNase I reaction Buffer:
200 mM Tris-HCl (pH 8.4), 20 mM MgCl2, 500 mM KCl

more protocols can be found on this page
http://www.protocol-online.org/prot/Molecu...tion/index.html

Thank you! I will give the Invotrogen protocol a try. I am guessing it is adaptable to other brands of Dnase I.

I would recommend putting 3mM CaCl2 into the DNAse I Buffer as well for optimal digestion