Hello,

It does seem that your PCR has not worked. The two bands that you see on the gel might be the two conformations of plasmids: open-circular (oc) and covalently closed circular (ccc) DNA and it is not uncoomon to see these two forms on the gel.

With no band in any of your vector+insert colonies, it seems to me that your PCR is a problem. If the primers were designed from the sequences within the insert, then you have no way to figure out the insert. But if the primers were designed from the flanking sequences of the insert, then your positive control of vector alone should have given you a band that must have the some size of the flanking sequences minus the expected band of 300 bp from the insert as it is not there in the vector alone plasmid. Now even this may not be easy to see on the gel, if your primers are really close to the insert and on amplifiction produce very short band of 10-20 or 30 bp, then it won't be clearly visible on the gel.

So, it all depends on yoru primer positions to trouble-shoot this.

Hope it helps,

SB

Hi Saadlee,

I would say you simply saw on gel your miniprep vector plasmid DNA: 300 ng are a lot, part will surely have degraded during the PCR reaction, but even if you had 50 ng left, you would see them on agarose gel. Circular (non'linearized) plasmid DNA normally has two forms, a so-called supercoiled that runs slightly below the linear plasmid (in your case 5 kb) and a nicked form, that is a relaxed circular form that has higher steric hindrance and runs much slower... probably around 8 kb apparent size fits...

Of course it means your PCR didn't work at all.

The only thing I see in your protocol the way you wrote it, is that you doubled the reaction buffer (5 ul of 10x in 25 ul... but often the buffer is 5x, so it might have only been typo on your side... Anyway if you did double it, you increased the salt to 100 mM instead of 50 and doubled the Mg2^ to 3 mM instead of the classical 1.5... This could have unpredictable results on the processivity of the enzyme). For the rest, the conditions are pretty much all right and PCR on a plasmid is not particularly tricky. What I can suggest is to check your primers, that they are not grossly wrong in terms of sequence or producing primer dimers. Secondly, for a PCR on a plasmid 10 ng of template are more than sufficient, too much template DNA can inhibit the Taq Pol.

Cheers.

Thanks a lot. If I use 36 cycles instead of 30 and 20 ng of DNA template, are they make any differences?

Other concentrations are:

10 mM dNTP = 0.5 ul, taq polymerase = 0.25 ul, 10uM primer (F and R) = 1 ul each, 10x buffer = 2.5 ul.

Hi again,

sorry I didn't see your answer... Normally on a plasmid 20 ng is plenty of template, 30 cycles will suffice.

Yes, your conditions are standard, and 0.4 uM primers should be enough to allow amplification of quite some template!

Keep in mind that colony PCR is tricky and often unreliable... If it works, fine, but if it doesen't don't despair, just grow some standard minipreps and extract your plasmid DNA... takes an extra night, but is reliable!

Good luck.