Hi Ronni,

Is there any chance that your colony PCR is giving false positives and you are cutting re-ligated vector? This happens quite often if you take the colonies directly instead of replicating them in a plate before doing the colony. The plate use in the transformation may carry enough “insert” to trick the PCR. Besides you mention that the colony PCR was carried out with the primers used to amplify the inserts. It would be better when you use a primer sitting on your ORF and other in the vector or in the next fragment you want to anneal.

Also make yourself 300% sure your vector is properly dephosphorylated. I recommend Antarctic phosphatase for this.

Hope it helps,

Hi Sebastian,

Thanks for you answer!

No chance of it being the vector, it too would digest with these enzymes. The thing is - basically any plasmid in the lab, from this reaction or a contamination from any other work, will cut one way or another with these enzymes. This is why this undigestable DNA is so perplexing to me.

The colony PCR was not carried out with the same primers that I used to amplify the inserts, it was done with a different set of primers, but later sequencing (with that different set of primers) always returns 'no reaction'.

The vector was not de-phosphorylated this time, but the same happened in my previous attempt in which it was de-phosphorylated properly. But anyway as I said, this strange DNA does not at all behave like my vector so I don't think it is it.

Would love to hear any other thoughts,

Ronni

Hi Ronni,

Thanks for the extra information.

I would still think that what you get is a sub-product of the original vector. maybe caused by excessive digestion/nicking, filling of the GAP and re-ligation. Either that or you got contamination in your digested vector vial with a smaller vector, more prone to re-ligation and not containing those restriction sites you use.

I would try reducing the reaction time to avoid the formation of sub-products (10-15 minutes).

The DNA you purify from the colony, have you tried to transform bacteria with it? I mean to see if it is a functional plasmid or just a part of one, lacking for instance the antibiotic resistance.

At last, and assuming your priority is to get the clone, I would recommend to do a overlapping PCR to join your 2 PCR fragments using the primers you already have (I can give you a protocol for that) and then you use the Gibson assembly to clone the assembled fragment into the vector. The reaction of a single insert with the plasmid should be more efficient.

Best,