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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.

-saeko-

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 95C, 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 37C 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 70C 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)

-Mischa Li-

Hi,  Mischa.

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

Saeko :)

-saeko-