plasmid preparation - (Nov/20/2008 )
I extracted plasmid from E. coli JM109 using QIAgen Miniprep Kit. But from the gel, I suspect that genomic DNA was not precipitated before loading the supernatant on the column, is that true? I conducted the experiments faithfully according to the manual. Can anyone give me any suggestion?Thanks in advance.
(Fome lane 2 to 5 are the plasmid prep lane)
it looks like you have too concentrated plasmids. try diluting 1:50 and run. Why would you want genomic DNA in your purified plasmids?
PCR with the primers for your plasmid and see if you have the plasmids there.
Dilute t he DNA to a lower level aas suggested. Then, digest some of the DNA with an enzyme that is unique in your plasmid. If you get linearised DNA, you just have bucketlaods of plasmid. It's actually doubtful that you have gDNA; it's the same size, and if you sheared the chromosome, you'd get a smear instead.
Are the 4 lanes meant to be the same plasmid, grown in the same cells?
If you followed the protocol, the gDNA (as well as proteins) would have been precipitated when you added the acetate (neutralisation buffer), assuming you mixed it correctly. Do you use the LyseBlue reagent? It's very good for seeing if you've completely mixed the sample.
You are fine. I am sure that's plasmid DNA.
Are the 4 lanes meant to be the same plasmid, grown in the same cells?
If you followed the protocol, the gDNA (as well as proteins) would have been precipitated when you added the acetate (neutralisation buffer), assuming you mixed it correctly. Do you use the LyseBlue reagent? It's very good for seeing if you've completely mixed the sample.
I did not use the LyseBlue. They are the same size because all the 4 lanes are the same plasmids with same inserts. This I can explain. But according to the marker besides, it indicates more likely to be the gDNA since they are over 10 kb. I don't know what is the weak bands just below the strong ones. They have similar length with my plasmids but I don't think they are plasmids.
PCR with the primers for your plasmid and see if you have the plasmids there.
I don't want the gDNA so I expect them to be precipitated before loading the column.
Than you, chessplayer. But I forgot to say that the marker besides indicates they are not the correct size. Do you think to concentrated plasmids will decrease the migrating rate on the gel?
I did an electrophoresis of the 50X dilution of my plasmids and it looks much nicer. But the size is still not the same as the expected ones. I guess it is due to the sepcial structure of the plasmids, circle or super-coil form will both effect the migration rate and make them different from the one of linear DNA. Is that the right explanation?
yea it has to do with the structure of the plasmid. u might have supercoiled, nicked and possibly linearized plasmid.
Dilute them and digest with a unique restriction enzyme.
that should tell you the next story.
(Fome lane 2 to 5 are the plasmid prep lane)
hELLO.
first, the quality of the preparation of your agarose gel is not good; you have two layers of agarose: try always to poor agarose gel one stroke.
second, i want to ask u what is the volume of you culture from which you harvested cells?