Posted 13 February 2009 - 07:43 PM
Posted 13 February 2009 - 11:19 PM
Posted 17 February 2009 - 06:39 AM
Sorry, I might be wrong, please correct me if so. Wouldn't we expect the A260/A230 Ratio be low if the salt concentration was high? I have been experiencing this prblem as well and I haven't been able to sort out what the hell is happening with similar A260/A230 Ratios (I've even got one at 20-something).
Posted 17 February 2009 - 06:52 PM
Posted 18 February 2009 - 10:45 AM
Had you checked your DNase I treated RNA on a gel? That would give you a visual confirmation (better than spec) if you have any RNA and DNA left. I don't understand why you need to do the DNase digest at 65C and why you would add EDTA to the DNase I reaction. DNase I works well at RT or 37*C and adding EDTA will remove the divalent ions required for DNase I activity. It makes no sense after doing DNase treatment without EDTA (correct way) and then you repeated it with EDTA (works poorly if any). Usually you add EDTA after the DNaseI reaction and then heat inactivate the DNase in the presence of EDTA to protect the RNA. I am puzzled why you would need to read OD of the cDNA reaction and there were tons of nucleotides (or maybe you had precipitated the cDNA?). It sounds you didn't know what you were doing buddie.