Distinguish uncut vs. cut plasmids on agarose gel - (Apr/28/2006 )

Hi All,

Am trying to check if my pcDNA3.1(+) which is 5.4kb is being linearized properly by AflII and also XhoI

When I digest and run it against uncut pcDNA3.1(+) this is what I get:

For either AflII or Xho I - 1 band running at 5.4kb

For uncut plasmid I see 4 bands - 1 faint band at 5.4kb and then band 2 is higher up - waaaay higher up, past the last molecular weight marker of 12kb and it's very bright and then I see bands 3 and 4 even higher than that

I figure that bands 2,3, 4 are nicked plasmid or some weird supercoiled version? But what is band 1 which is running at the same size as the plasmid I cut with AflII or XhoI? Can I assume my plasmid has been linearized properly?

Thanks!

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can you atach the gel picture please?
how long is your digest? (recommend at least 1h30, 2h is better)
how much plasmid do you digest? is the dna/enzyme ratio not too high?

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The supercoiled version of the plasmid would run smaller than the liearized, as would the nicked...

Could this be chromosomal DNA? How did you isolate your plasmid?

I suppose this might also be plasmid dimers -- what host strain were they isolated from?

A picture of the gel would help a lot...

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HI

Ok, I've got a JPEG image and I've attached it as a file attachment - hope you can see it? THe image file isn't too great but there is a faint band at 5.4kb for the uncut plasmid.


The plasmid is pcDNA3.1(+) commercially bought from Invitrogen - only thing I did was transform it into some DH5alpha cells and grow more up and recovered it with Qiagens' mini prep kit.

Am digesting for 1 hour 30minutes to 2 hours at 37oC.
Thanks!

E-star

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First of all, I think your gel percentage is too high. For plasmids, I usually use 0.8%.
Next, you might want to re-run with larger markers. Do you have any lambda digests?

As for the reactions,it looks as though your undigested plasmid may be dimerised, or something, but you cannot say, because it's waay too big for the markers you have run.

AflII looks OK, but the Xho looks slightly undigested. What are you planning? If it is for cloning, you might want to simply gel-extract and do the second reaction, if you're planning a double-digested insert.

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It's a 1% gel and the standards I use should be high molecular weight enough to handle my plasmid - the top band is suppose to be 12Kb and my plasmid is only 5.4Kb

Is it the gel percentage then?

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The gel percentage is ok I think. I use 0.8 or 1 % agarose. As I mentioned somewhere else I also work with pCDNA3.1 and usually you see 3 bands for the undigested vector. One at approximately 3 kb (supercoiled; quite bright), one at 5,4 kb and a very faint band somewhere higher.
As your vector is more or less properly cut with the two enzymes (which doesn`t mean they work fine in a double digest though) I would simply go on with the transformation or try to isolate the plasmid again very carefully (f.ex. not vortexing it).
The digestion seems to separate the structures present in the undigested vector prep.

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hi
for me the digestion seems fine at least for one enzyme (regarding the fact the short DNA fragment is not viewable).
To evaluate the quality of digest, you can transform little part of your prep.
Then you can dephosphorylate vector if you want more secure.
Then go for cloning.

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I agree -- I would not get too hung up on the appearance of your uncut vector lane, or the spurious bands in the cut vector lane -- as I mentioned before, I'll bet these are some kind of weird plasmid dimers.

Does Invitrogen technical support have any explanation or insight?

Were I you, I'd just go for the cloning with an appropriately restricted band recovered from a gel...

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Yep - I've gone ahead and done what homebrew suggested - cut out the band from the gel - so will go ahead with the cloning and see what happens.

Have also ordered some SAP and CIP too, just in case!

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