How can I purify these nonautoclavables? And do I need to? - (Sep/16/2004 )
I am trying to narrow down a problem with plasmid DNA. If you have read my other posts they are on the same topic. I think there may be a nuclease or other contaminant in my isolation materials which keeps ending up in the final liquid with the plasmid DNA. To prevent this how should I purify these non-autoclavable things? My aim is to clear them all from suspicion of contaminant. Let me know too if they would even be likely to carry-over contamination.
I can autoclave or filter sterilize everything else in the procedure. Ordering new of any of these can be done too if neccessary.
I would have thought that the best and most sure way would be to buy new reagents, and make sure that all your tips, tubes and anything, including pipettes, that comes into contact with the reagents is clean before starting again. I would certainly do this with the cheaper reagents such as phenol, ethanol, chlorofom and EtBr.
You could try to re-purify the enzymes using an affinity column, though this may be more trouble than it is worth.
Best of luck
RNase A (?) is frequently contaminted with DNase I. Heat the RNase prep 100°C. for 10 minutes to denature DNase I. Incubate 10 µL RNase A with 1 µg pBR322 cut with Msp I or Hinf I at 37°C. overnight. Check for residual DNase activity via PAGE. If residual DNase I activity exists, repeat the boiling step. Recheck for contaminating DNase I. If residual activity remains, discard the prep and start over, as RNase A activity is inhibited if the process is repeated a 3rd time. Note that you can make another attempt to prepare clean RNase A from the same bottle with reasonable luck. Aliquot and store at –20°C.
I would assume that your other components are RNase and DNase free.