How much PCR do I need for cloning? - (Dec/05/2006 )

Hi!

I was wondering how much of the PCR product do you need for cloning. I have my PCR ready for digestion but the band was quite weak so I was wondering that maybe it is not enough..

Does anybody have any idea?

j

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You mean for ligation?

Not too much, some nanograms of PCR product will do most likely.

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Vairus is right, I usually do PCR with 20ng of DNA for cloning. Then as long as u see a band of product, the PCR product should be more than 50ng.
It also helps to do ur digest in a small volume so that the DNA is more concentrated.
good luck

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I mean for the whole procedure: digestion, gel purification, ligation- how much of the PCR do you need?

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It depends. (A most unsatisfactory answer.)

It depends on
1- on your technique. How good are you?
2- the size of your PCR product... 400bp verse 10kb. You need less product mass for smaller products. As the same mass give more mol of products for smaller molecules compare to larger molecules
3 - and your construction 'style'. Do you like working with large quantites of DNA (buffering against losses) or small quantites (enzymatic manipulation are quick)

So how much do you need?

Well, you probably only need ~500ng to get by.

However, in your situation I would recommend using 1ug to 2ug of PCR products. That would be plenty of DNA to buffer against losses. And any missadventures that might befall.

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I agree,
If the band is weak, I would run a large volume of my PCR to have product enough. If not, there is a risk of have nothing at the end .
Bye the way, medchemgirl, We are able to see 3-6 ng of DNA in an agarosa gel.

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