Basic Question Restriction Digest - (May/30/2006 )

I've done Restriction Digestion of my plasmid with EcoRI and I've obtained this picture:



I've done Restriction Digestion for 1hour using a specific buffer (buffer H), BSA and EcoRI restriction enzyme. My intital plasmid is 1ug.
The result is fine, but as you can see, my plasmid shows a higher concentration than my insert. Is this normal?
Could anyone tell me what I can do to increase my insert concentration?

I have three ideas in mind:

1- Increase the incubation time.
2- My enzyme is out of date, so increase the amount of enzyme (supposing that the efficiency has decreased).
3- Purchase a new one.

To be honest, I don't think I will need to do often restricion digestion. What do you recommend me?
Cheers

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the upper band is more intense than the lower band because there is more bp in the upper band than in the lower : there is more bp in 1 nmole of the vector than in 1 nmol of your insert. then the band of the vector is more intense than the band of your insert.

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hi
i agree with missele, but maybe you can do a 2-4 hour digestion to be sure. I'm surprised that you don't have any piece of uncut or single cutted vector...

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I agree with both missele and fred.

U could increase your DNA from 1ug to 5 ug, u will get 5x more insert.

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hi fred...should there always be a single cut or uncut vector visible....

increasing the dna amount will certainly increase the amount of insert, but the difference in the intensity on the gel between vector and insert will remain...

viv

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Thank you for all the comments.

I didn't think about this issue of the single cutted vector. Might it be because the gel didn't run long enough and uncutted and cutted plasmid still together? (only 30min and you can see that the marker's bands are together on the top).

I think I will try again leaving the incubation longer and running longer the gel.

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If I were doing it, I would also load some non cut plasmid on the gel. it's easier to check if it is cut when you can compare cut and non cut.

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i mentionned that point due to the fact it was "only" a 1h digestion and in general, i consider it's a short time and some uncutted or single cutted vector would show up. Always,not for sure, but i was surprised, that's why i putted it in my reply.

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gsamsa, your question was answered in Misselle's first post, I think

you do not have 'more' vector than insert, you likely have the same number of molecules in each band...but insert is short and vector is long, so the vector band will have more length for the etbr to intercalate and will appear brighter

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That's right. Firstly, Ithough that if I've loaded a ug DNA plasmid I should get around 1ug (maybe 0.8ug because of enzyme efficiency) of the lower band (based on stoichiometry). But just now I realised that is a weight relationship, so I suppose that if my plasmid with insert is ~6.5kb and the insert 0.8kb I should get 120ng at most.

This are my calculations:

1000ng plasmid with insert *6.5kb= x ng *0.8kb.

Am I right about this?

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