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RNase treatment of genomic DNA

Comments

• prepare 37°C water bath in foam plastic box;



estimate DNA quality on 1.5% agarose gel (load ~0.2-0.6µg DNA);
prepare reaction (up to 20% of RNA):
DNA, / 10x RNase ONE buffer RNase ONE, 10u/µl H2O
37°C, 1h;
check presence of RNA on 1.5% agarose gel, store reaction in ice during electrophoresis (load ~0.2-0.6µg DNA; it is desirable to run control/untreated DNA);
spin-column purification

• add 5xV of PB:
• column type:
       <10µg: QIAquick
       10-20µg: QIAprep
snap freeze in liquid nitrogen and store at -20°C;

Notes

Small RNA fragments are hardly visible on agarose gel, but the molar quantity may be high and they may inhibit polynucleotide kinase and polymerases.

Store RNase in separate freezer in special box.

Phenol/Chlorophorm purification may be performed for >20µg of DNA.

It is possible to selectively remove single-stranded RNA and purify only double-stranded DNA on silica column (protocol on Russian).

It is possible to shear DNA by ultrasound directly after RNase treatment w/o purification.

It is possible (but not desirable) snap freeze DNA in liquid nitrogen and store at -20°C after RNase treatment w/o purification.