Subtle change in insert size after cloning - (Nov/19/2014 )

Hello!

I'm cloning a 340bp insert in pGem-T Easy vector. After miniprep and digesting with EcoRI procedure, I run the agarose gel and see the right band for the plasmid (around 3000bp), but my insert is always on 500-600bp. I already double-checked the insert before ligation and transformation through agarose and by measuring D.O. again, used other kits, changed protocols for transforming, made new reagents used for it, but the final result is always the same. 
I already sequenced it, and got a lucipherase gene when I'm expecting a fragment of an antibody gene, but when I PCRed the product of miniprep, there was a good amplification of my targeted gene.
What could be happening?

The most likely scenario is that you aren't amplifying what you think you are.

bob1 on Wed Nov 19 20:55:44 2014 said:

The most likely scenario is that you aren't amplifying what you think you are.

Even considering the specifity of my primers at the PCR and all?

Yes - you could easily have mixed one or both of the primers, or be using them on the wrong template. The primers could also potentially amplify more than one site on the plasmid - it usually pays to check this when designing.

bob1 on Thu Nov 20 07:26:53 2014 said:

Yes - you could easily have mixed one or both of the primers, or be using them on the wrong template. The primers could also potentially amplify more than one site on the plasmid - it usually pays to check this when designing.

Actually, I didn't start from any plasmid; I extracted RNA from my cells, and then getting the cDNA I made the PCR using primers for a conserved region of IgG genes, purified it from the agarose gel and inserted it in the plasmidial vector. Anyway, I just checked the primers as you said, their very well identified, I'm pretty sure I used the right ones (specially because I made it more than once until I get a good gel purifying technique, haha)

Thing is that I got 3 similar lineages of IgG secreting hybridomas, and my lab group has always amplified the genes that interest us with these primers when talking about antibodies. I'm trying to understand how can it be possible that the agarose tells me 300bp before cloning, and after digestion the same DNA is around 500-600bp... Control tells me I'm using the right RE. Should I start it all again from RNA extraction???

Oh - I see, I thought you were amplifying off the plasmid and getting the wrong size for the second part of your original post.

According to Addgene pGEM-T has two sites that will add an extra 90 bp to the insert when digested - this would take the product to 430 bp...

Well, sequencing told me 585 bp twice already hahaha
I'm gonna keep trying, but thanks for answering me!