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

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Did you visualize your DNA with Ethidium Bromide? If that's the case, your insert band might appear weaker because the dye migrates in the opposite direction as the DNA, i.e. the dye front might be above your insert and below the vector.

If the plasmid wasn't properly cut, you should see two vector bands (unless you haven't let the gel migrate long enough).


QUOTE (gsamsa @ May 30 2006, 05:42 PM)
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2- My enzyme is out of date, so increase the amount of enzyme (supposing that the efficiency has decreased).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?


QUOTE (gsamsa @ Jun 1 2006, 01:03 PM)
QUOTE (aimikins @ May 31 2006, 09:51 PM)

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

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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