Smear on gel after MiniPrep - Is this RNA? (Oct/23/2006 )
I did several ligations, picked colonies and did a mini prep. After applying (EcoRI) digests of the mini probes on a gel (1% Agarose) I couldn't identify a band at 280 bps because there was lots of smear. Is this RNA?? I think so, but would like to have a second opinion. Should I use RNAse after ligation before digestion?
Was RNAse used during the miniprep extraction? The absense of RNAse would well explain the strong RNA like band.
Anyhow I would guess that the bands you are seeing is RNA
this really looks like RNA.
Usually I use RNase in solution I (digestion)
I didn't use RNAse in the Miniprep. But then again I never did so and never encountered any problems...
I checked whether we have RNAse. We only have a solution of 100mg/ml and no one knows what the solvent is...
I will go crazy...
as far as i know, you are using QIAgen spin miniprep kit. u need to add RNAse into solution I (resuspension buffer) in the kit. that way, you will get rid off RNAs.
Actually I don't have a kit for minis. I make the buffers by myself. And the RNase we have has a concentration of 100mg/ml (!!!) which is wy too much. In what am I supposed to dilute this?
What should the final concentration of RNAse be? Suppose I have the pellets of the minis dissolved in 30 µl. How much RNAse should I add?
RNAse should be used at final 100µg/ml for 15-30' at37°C
then treat with proteinase K if you matter of subsequent aplications, followed by phenol chloroform
The RNase we use is a 10X stock at 100ug/mL. So, if you have a 20ul digestion, you would want 2ul of RNase. I hope this helps!
As I have mentioned we only have RNAse A 100mg/ml. In what am I supposed to dilute it in order to add to my "DNA in TE" samples. I don't want to completely dissolve the DNA obtained from mini prep in a RNAse-containing buffer, to avoid the phenol/chloroform step in case of required subsequent transformations...