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Agarose gel electrophoresis - why does my bp not add up?

Will be as brief as I can! - any help would be appreciated.

used 3 lanes on my gel and ran in each:

lane 1: hyperladder (linear fragments)
lane 2: EcoR1 digested recombinant pUC18 (size of insert unknown)
lane 3: undigested recombinant pUC18 plasmid

Results obtained from gel and standard curve:

lane 2: 2 bands apparent - determined as 3890 and 257 bp.
lane 3: 3 bands apparent - linear band determined as 5011bp

A pUC18 plasmid (without insert) has 2687bp so from the results obtained from lane 3 this would suggest the insert is 2324bp. My results from lane 2 don't confirm this at all though!!

The largest fragment band of digested pUC18 (lane 2) migrated further than the linear undigested pUC18 in lane 3 and almost as far as the band which represents the undigested plasmid in its supercoiled form. I don't understand why????

I realise that this method of determining the size in bp of DNA is not very acurate but this doesn't explain why the digested fragment migrated further than the undigested pUC18.

If you can help at all I'd be really grateful - many thanks


Solution Preview

Don't forgot the fragments in lane 2 are from the pUC18 plasmid with an insert. I wasn't 100% sure from how you asked your question whether or not that was clear to you.

So the biggest question here is do you know anything about your insert? Normally when I am cloning into a plasmid, I know alot about the insert, so I would know where EcoRI would cut it. EcoRI cuts pUC18 only once in the multiple cloning site (according to the map I am looking at, the digesting with ...

Solution Summary

The expert determines Agarose gel electrophoresis is determined. Why does the bp not add up is analyzed. The results obtained from gel and standard curves is given.