I did a Birnboim and Boly plasmid Prep for my plasmid and insert which are grown on BL21. After that, I digested them with my restriction enzymes and run the samples on the gel. What I got was a huge blob of band and multiple bands on each lane. I tried adding RNase as I suspected it was contaminated with BL21 RNAs. The blob disappeared mildly but so does the rest of the bands except for one lane. Why does this happen?
Dear Liyana, It looks as if you have isolated a substantial amount of RNA in your Birnboim and Doly alkaline lysis plasmid preparation. The technique does not distinguish between RNA and DNA, so a RNAse step is usually required if you want to prepare a large amount of plasmid DNA for further work. You will also probably find that you have some chromosomal DNA in the preparation too, though usually this should not interfere with normal restriction analysis of the plasmid DNA. Regards, Andrew
Dear Liyana, Also note that most commercial RNAse preps may contain contaminating DNAse. You may want to place your capped tube of RNAse in a boiling water bath for about 5 min., then take it out and let it cool slowly to room temperature. Any contaminating DNAse will be irreversibly denatured by the heating, but your RNAse should survive (use RNAse A). Also, to prevent other contaminating DNAses from being active during RNAse digestion, include up to 10 mM EDTA in your RNase digestion reaction.
Right. Thanks David and Andrew for the suggestions! I tried doing my RNase reaction in a water bath at 37C and most of the white blob disappeared but so does my other bands except for one lane.
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