dephosphate the digested vector - (Jul/09/2005 )

Hello.

I am thinking about a silly problem: when should i dephosphate the double-digested vector? if i don't dephosphate it, how it affects the ligation? Should i dephosphate the fragments which i am going to insert into the vector?

In my experiments, i usually don't dephosphate the vector and fragment, but the efficiency is not so high.

Please give me your valuable suggestion on whether or not i should dephosphate the fragments before ligation.

Looking forward to your reply!

Thanks.

sincerely

boychina

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Ideally a double digested vector (i.e. digested by two different enzymes) should not require dephosphorylation. However, if not all your plasmids are digested by both enzymes (say a small % digested by only one), then the dephosphorylation step will reduce your background religation (vector only religation). Some say CIP is damaging in large quantities and should not be used unless you need to. But I dephosphorylate anyway and seem to get inserts.

I dephosphrylate after gel extraction of digested vector - and then PURIFY after dephosphylation (essential to remove excess CIP)

I think inserts are not usually dephosphorylated since you need a source of Phosphate to power the religation - so only the vector is dephosphorylated. Hope I've helped.

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I recently experienced a "bamHI-EcoRI" self-ligation (hence a GATC-AATT ligation) in my vector in 6 out of 10 cases (on my positive plate this was, I had only one colony on my negative control). So I would go for defosforylation, or check if your enzymes give really completely different overhangs.

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Vairus: Have you sequenced these, and determined that ligation happened between BamHI and EcoRI sites, or is this just that you have some empty clones? Empty clones could result from cutting with a single enzyme and religation, for instance. Since I'm trying to optimize ligations, I really want to understand failures like this if they are happening.

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I didn't sequence, but when doing restriction analysis, the fragments I got could only be explained by vector self-ligation. (I cut my vector with bamhI and EcorI, I cut again with NheI as this cuts only in my original insert and by this I could prevent self-ligation without gel-purification and then added my insert that was also BamHI-EcorI cut)

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