extract plasmids from transfected eukaryotic cells - (Sep/19/2011 )
for my research on oxidative stress I need to conduct an experiment that sounds a bit strange in the first place:
I want to transiently transfect and eukaryotic cell line, expose it to some environmental stress conditions and afterwards regain the transfected plasmids from the cells for transformation of bacterial cells and subsequent analysis. I am wondering what would be the best way to prepare the plasmids from the eukarytic cells. I thought of TriZOL lysis, which always results in plasmids in the "RNA" containing upper phase as i know from qPCR studies, then ethanol precipitation and then RNAse digest or, instead of RNAse, a chloroform-phenol-extraction with basic phenol pH8 which should give a good separation of the remaining plasmids from the unwanted RNA. Or do you think it more feasible to use something like a miniprep kit?
Has anybody done this before and wants to share his/her knowledge with me? Any help is appreciated.
By far the easiest would be to use PCR to amplify the plasmid. Another alternative would be the use of rolling circle amplification to preferentially amplify small circular DNA. With modern enzymes such as Phusion, PCR errors are relatively unlikely.
unfortunately PCR is not an ideal option. I want to subject the plasmids from the transformed bacteria to parrallel sequencing to see if the plasmids aquired any mutation during their time in the eukaryotic cell. Any PCR error would be misleading in the experiment, and since i expect mutation to be a low frequency event i would rather not test my chances even with a modern polymerase if i can avoid it. But anyway, thanks for your suggestion!
I don't think that plasmids typically get replicated in eukaryotic cells (though I do seem to recall this happening in some cell lines), so the chance of mutation is quite low, I would have thought. The other types of mutation (e.g. crosslinking, ds and ss breaks etc.), you won't detect by rescuing the plasmids and growing them up again.
I want to detect a specific mutation pattern that is caused by oxidative DNA modifications. For my initial experiments i will use HEK293T and COS7 cells, both carrying the SV40 large T antigen, which should replicate any vector with a SV40 promoter, at least that is what i read. For my further studies in more physiological cell lines i thought of a plasmid that overexpresses the large T antigen and therefore is kind of self-replicating that carries my specific detector sequence. I think that should work, do you?
Just found something called Hirt Preparation:
HIRT DNA PREP FOR ANIMAL CELLS
A method to prepare episomal DNA from animal cells (transfected vectors,
Hirt lysis buffer: 0.6% SDS, 10 mM EDTA pH 7.5
5 M NaCl
RNase A: 2 mg/ml
Phenol, saturated with 0.1 M Tris pH 7.4
5 M Sodium acetate pH 6.0
1. Wash 10 cm confluent plates with 1 x PBS at room temperature.
2. Add 2.0 ml. of Hirt lysis buffer. Let sit at room temperature 0-20 min.
3. Scrape lysate into 12 ml Falcon tubes. Pour into SW50.7 polyallomer tubes.
4. Add 1/4 volume 5 M NaCl, cover top with parafilm.
5. Store at 4°C for 8-20 hrs.
6. Spin at 4°C 18,000 rpm/40 min.
7. Pour supernatant into 12 ml Falcon tube; add 25x of RNase A (2 mg/ml). Incubate at
37°C for 60 min.
8. Phenol extract 2x.
Ether extract 1x.
9. Add sodium acetate to 100 mM;
Add 5 ml isopropanol, store overnight at -20°C.
10. Spin at 10 K for 30 min.
11. Rinse pellet with ice cold 70% ethanol; invert, air dry, resuspend pellet in TE.
I think i will give this a try...
Looks pretty good, fairly standard DNA extraction. You might need plasmids with the SV-40 origin of replication rather than the promoter!
I have been thinking about this a bit, as some of my plasmids are episomal, and I get low transfection rates in some of my cell lines, so was thinking about whether maintenance was an issue...
Do you have any original references for the episomal replication by T-Ag? I've only been able to find papers that talk about it in the intro without references.