I amplified my DNA insert for cloning and confirmed the product by agarose gel electrophoresis. The gel showed a clear, single band at approximately 1.4 kb, which matches the expected size of my insert (1.36 kb).
Following this, I performed A-tailing on the PCR product and used it for TA cloning into the pGEM vector. After transformation and colony PCR screening, I ran the colony PCR products on a gel and observed a single, clear band between 2–3 kb, which was unexpected.
To troubleshoot, I went back and ran the A-tailed product on a gel. Surprisingly, it appeared as a single band larger than 1.5 kb—larger than the original PCR product.
Could you help me understand what might be causing these size discrepancies? Is it normal for A-tailing or TA cloning to cause such shifts in apparent size?
Hi! I guess the only thing I can think of is: are you sure you're not amplifying the vector rather than the insert in your colony PCR? If you can send it for sequencing, I'd do so, but my suspicion is that you're amplifying the vector pGEM (which is of around 3 kb) and not the insert. Let us know if you checke the sequence in the future and good luck!
How did you do the A tailing? maybe you added too much A on your insert.... colony PCR has lot of drawbacks; I prefer plasmid minipreps analyzed by restriction enzyme followed by sequencing; does not take much more time than colony PCR
Didier Poncet I performed both enzyme digestion and colony PCR. After enzyme digestion, I observed a single strong band slightly above 3 kb, which I suspect corresponds to the pGEM vector. Colony PCR using gene-specific primers yielded a single strong band slightly above 2 kb. However, when using vector-specific (M13) primers for colony PCR, I saw two bands: one slightly over 2 kb and another around 600 bp. This unexpected result has left me quite confused.
Natalia Salazar-Quiroz Yes. I sent the sample for sequencing. Since pGEM is ~3 kb and my PCR band was slightly over 2 kb, I didn’t expect the amplified product to be the vector itself. However, the sequencing data revealed that most of the 'insert' was actually a fragment of the vector. What’s more confusing is that the sequence appeared to originate from the reverse primer site and proceeded in a counter-clockwise direction. Essentially, the 'insert' sequence matched a region of the vector upstream of the forward primer, with a gap between this fragment and the expected forward primer binding site—making the result even more perplexing.
@Xuebi I see, but just to clarify, this is the result from the colony PCR, right? If you do a miniprep for the plasmid and send that for sequencing, do you get the desired insert size? If that's the case then the extra bases come from an artifact of the colony PCR and wouldn't bother troubleshooting that. If your mini is the desired plasmid, then stick to that
puzzling!! but you have the answer= your clones are not OK !! for TA cloning after amplification with a proof reading polymerase (pfu or Q5) i use to make 2 more PCR cylcle with Taq polymerase (just to add some A at the ends) and use commercial vector kit with blue/white screening...
Hi ! The discrepancies you’re seeing are well within the range of normal behavior for cloning workflows involving TA cloning and colony PCR using vector primers. These shifts in gel mobility are typically artifacts of the method, not actual errors.
Your cloning is likely successful you're now at the stage where you should verify with restriction digestion or sequencing to move forward confidently.
If you'd like help selecting restriction enzymes or designing sequencing primers, feel free to ask.
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