plasmid mini-prep from E.coli - smeared bands (Feb/13/2002 )

Hi.  I have a little problem with mini-prep of 3.5kbp plasmid from E.coli. The plasmid carries my gene of interest(after subcloning).  
When I did mini-prep a few months ago, the bands on the 0.7% agarose gel looked fine(very clear and not smeared, no RNA appeared on the gel). And then I do the mini-prep from the exactly the same culture(glycerol stock) now, the bands look very smeared and I can see a band in the low molecular weight (I think it is RNA, which I did not observe a few months ago).  

I did the exactly the same mini-prep from the same culture, but why they look different on the gel?
I do RNaseA digestion during the mini-prep, but why I still see the band which seems like RNA??? I thought RNase is stable and I did not care much about it(it worked fine before).

What do you think is the problem here? the stock, procedure, or one of the reagents?
One thing I changed from before is agarose.  I used to use different company's agarose. But it is hard to imagine the agarose can make this difference.

Thank you in advance.

Hi,

smeared bands can have several causes. I never had a problem with the glycerol stock! But I had a lot of problems with solutions and DNAase! Especially when several persons used my solutions and buffers (and I did not know it)! Now, I am working alone with my solutions and stocks in my lab and the problems are gone!
First of all: inactive RNAase let residual RNA in the DNA-preparation (smaller then 1 kB) but RNAase makes NO smeared band (3.5 kBp)!
For an "expertise" I need more infos but try to check some important things:
1)wrong electrophoresis: TEA-buffer (pH!, salt concentration); loading-buffer (do not heat it!)
Control: load in the slot next your plasmid-DNA a "surely good" DNA e.g. commercial 1-kb-ladder
If the "surely good" DNA is smeared too, change the appropriate buffer (maybe agarose too, but I do not believe really that ALL your agarose is SO bad)
If the "surely good" DNA is OK but not plasmid-DNA check in next step

2)DNAase-activity in your RNAase, water, buffer or restriction enzyme (if you cut before electrophoresis)
Control: treatment of "surely good" DNA with your RNAase (water, buffer...); for this control do not cut the plasmid-DNA before electrophoresis
If the "surely good" DNA is smeared too, incubate your RNAase for 15 min at 95°C, change the appropriate Buffer or water
If the "surely good" DNA is OK but not plasmid-DNA check in next step

3) DNAase-activity in your plasmid-DNA
Control: incubate your plasmid-DNA as it is at 37°C 8-12 h, put  each 2. hour an aliquot (~100ng) in a separate tube and freeze it immediately (do not forget the 0-probe without incubation!); incubate all the 2-hour-probes for 15 minutes at 70°C and check them via electrophoresis. If the smearing increases with the incubation time, DNAase did it!
Solution: treatment of plasmid-DNA with proteinase K, purify and check via electrophoresis
4)residual genomic DNA from the host in your plasmid-DNA (very often!)
Solution: better preparation of plasmid-DNA (try commercial plasmid-DNA-isolation-Kit)

Remeber: you never make EXACTLY THE SAME preparation!

Good luck!
M.L.

(Edited by Mischa Li at 2:13 am on Feb. 14, 2001)

Hi,  Mischa.

Thank you for your quick responce.
I will check some points you suggested.

Saeko :)