double digestion - (Jun/28/2007 )

Hey everyone,
I have some general questions about digesting a vector for cloning. So the protocol used in the lab to digest a vector for cloning is as follows.
1)digest with first enzyme
2)gel purify
3)digest with 2nd enzyme
4) dephosphorylate
5)column purify and stored at -20 until needed

I wanted to ask if gel purifying at step 2 is needed, can I not gel purify and just column purify and digest with the 2nd enzyme.

Also, from the above protocol, 1uL is enough for cloning ( I don’t bother measuring the concentration). What is the usual vector:insert ration that people use for cloning?

One more question, I don’t think column purifying removes the orginal insert that was cut from the plasmid. Why doesn’t it religate more often than the new insert you want to put into the vector? (does this question make sense?)

Thanks

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in my opinion, you should do the two digestions before doing the gel (or even doing two times a gel!), but sometimes buffers are compatible even if they come from another firm, so if it's the case you can digest with both enzymes together and then make a gel to purify.

I don't think the column remove any insert (even if I know we use High Pure from Roche and we observe a loss of shorts fragments).
That's all I can say

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Unless there is a major incompetability issues. Most restriction enzymes can be made to work in a double digest. And yes, a column purifying would also remove the first enzyme.




Ratio commonly in use are 1:1, 1:3, 1:5 and 1:10 vector:insert. This is not a law and you can use whatever ratio that works. However, the ratios use are ratios of molecules of vector to molecules or insert. Not ratio of volume. Different fragments can have wildly different concentrations due to avalability of starting material, processing loses and accidental loses. Unless you are confident that your insert is in abundance, you should check the DNA concentration.




Who says it doesn't? The vector of insert used to sit in must be removed. I believe the above protocol is not a general protocol.

1)double digest DNA if possible
(if not do sucessive digests, starting with the enzyme that uses the low salt buffer. NEB buffer 1,2 and 4 are low salt and buffer 3 is high salt)

2)gel purify. Excise the desire DNA fragment from the gel matrix and extract

3)find out DNA concentration

4) dephosphorylate. (beware overdephosphorylation will damage DNA ends, rendering DNA unligatable. May sometimes be trouble then it is worth)

5) stored at -20 until needed

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perseneblue is right. As for the column purification, it depends on the size of the insert. For normal vector, the insert is usually small, of course it will be removed. If you are cutting out your insert from another construct to reuse the vector (if the restriction sites can be used), then gel purificatiion is recommended. Even so, if you use column in that case, remember the ratio of ligation that you are going to use. The cut vector and old insert should be significantly less than the amount of insert that you want to put in, right? Do the math then. That is why we need positive and negative controls and need to screen our colonies later, isn't it?

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What is the purpose of gel purifying? Is it even necessary?

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to make vector or insert for ligation purpose you should make your strategy by your own way. the steps that you mentioned could not be the fixed strategy to make a vector. its steps would change according to what enzyme do you want to use, do u want to make it blunt or not, which fragment do you want to gel purify etc etc. if you digest your vector with 2 different enzyme you dont need to dephosphorylate it........so just think what you want to do and make your own steps

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Gel purification is important to remove other unwanted fragments.

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