PCR and Digest cleanup before ligation. - A shortcut by avoiding gel purification. (May/29/2007 )

No, I meant if it's a Nanodrop don't bother quantifying a cleaned fragment. I never get anything above 20ng/ul, and usually it's more like 6ng/ul. Running it on a gel shows a concentration of 50-100ng/ul. Running pure water through a silica column gives a funny reading, so I'm pretty sure the column leaves a residue which confuses the Nanodrop. Blanking the nanodrop with this residue doesn't seem to help.
-This is based on my experience with one Nanodrop machine
-I haven't compared it to an old fashioned spectrophotometer. Maybe it has the same problem too!

This is just from my experience (and those of my colleagues) so I'd like to hear your comments.

Alex, yes, you should do a cleanup on the vector digestion. Normally you would run that on a gel and cut out the large fragment, but in your case because the bit you're removing is so small you can just put it straight through the cleanup kit.

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Hi,

I think it's better to gel isolate your restricted vector DNA rather than purify it using QIAGEN's enzymatic reaction cleanup kit. The vector might not be 100 % restricted and residual uncut plasmids will overwhelm your plate later, when you do ligation and transformation. It happened to me and there'll be headache trying to PCR screen the colonies later.

If you want to skip gel isolation and purification of your restricted PCR product, it's ok I guess as the chipped off DNA at both ends are normally too small to be trapped by the silica gel filter column.

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True, but I think that a 2hr digestion with NEB enzymes will digest most of the vector DNA. I'll definitely run a positive control... I let you know if I get a high number of false positives or not.

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But not denatured plasmid DNA. No restriction enzyme will cuts those things. Denatured plasmid will remain unharmed and will still remains fully transformable, giving rise to future headaches.

Denatured plasmid is formed when a plasmid is exposed to an alkaline solution. The longer the contact, the more plasmid molecules are denatured. (Yes it is a balance, too short and the NaOH/SDS won't lyse all the bacteria, too long and too much plasmid DNA is converted into denatured plasmid). Denatured plasmid is the mystery 4th band that, on the rare occassion, one will see when running raw plasmid DNA. And if one where to cut the prep, this rather fast moving 4th band doesn't dissappear but remains.

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That's interesting...
Isn't the idea with the miniprep that the plasmid will re-nature when you add the neutralisation buffer (acetic acid at pH 5.5) while genomic DNA doesn't? Or is that process not perfect?

And mystery 4th band from raw plasmid? I thought it's just 2 bands, supercoiled and open-coil, at least that's the way I was taught, and that's what I tend to see... so what's the 3rd band?

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Freakin A, I hate that there are so many bands when I run gels.... but I think this is only the case with uncut ("raw") plasmid. The third band... wouldn't that be nicked plasmid? I believe the order of bands would be, from fastest to slowest:

1. super-coiled
2. open-coiled, linearized
3. nicked
4. that mysterious fourth band perneseblue is talking about. so mysterious. oooh.


By the way, I plated like 12 ligations I did during the week (using two vectors). Strange results.... the negative control (cut vector) plates had 8-13 colonies, the positive control (uncut vector) was a lawn, and the ligations had over 500 colonies. I have never had a transformation give me over 500 colonies. Anyway, the vector was definitely cut pretty well (only 8-13 colonies on negative control), but I bet that the sequence cut out from the MCS of the vector was not purified from the restriction digest and the vector recircularized (thus the high number of colonies on the ligation plate)... any thoughts?

I'll do some minipreps next week and let you guys know what's up.

Peace, fellow researchers.

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