Well, I would consider ~29bp of overhang to be massive. Even if your complementary DNA is around 10 bp, primer is now of length ~39bp. This will likely increase chances of formation of secondary structures in and among primers leading to reduced annealing. Furthermore, such massive overhang would lead to instability in annealing during initial cycles. Technically, if first few cycles are even moderately successful, latter cycles should have no problems (given primers do not form very stable secondary structures), but quite honestly, I don't see it to be likely.

You could check for any secondary structures in and among your primers here: https://eu.idtdna.com/pages/scitools

In my opinion, however, you would be better off using a vector which already has a his tag. Alternatively, you could also try attaching his-tag to your vector using biobricks method prior to ligating your insert but it is also rather complicated.

Hi All,

Thanks for the answer. I have managed to overcome the issue by using another bacterial expression system which has his tag in the vector.I shall be using it as a template of my PCR, thus avoiding the long overhang problem.

I agree that cloning into a different vector that already has a hexahistidine tag might be more prudent (e.g. pET24, pET28, pTrcHis, etc.). There are cheap versions of these on AddGene (addgene.org/jack_griffith).

However, I realize that sometimes there are reasons people want to stay in their current vector (e.g. comparability with previous experiments).

I have gotten away with using some rather large overhangs when cloning in adapter sites for difficult cloning projects - I suspect youy plan could work. If you get PCR product of the correct mass at usable quantities without using a high number of cycles, it will be no more likely to contain an error than any other PCR reaction.

When using long overhangs I've often made efforts to make the overhang partially complimentary to the region adjacent to the region of perfect homology (e.g. modifying wobble bases, chaning to other codons) to improve annealing in the first few cycles. This may not be possible or prudent in your case. For instance using a low-frequency codon might affect expression of the transgene.

You may need to optimize the reaction conditions to get good production of the desired product (e.g. changing [Primer], [Template], polymerase, annealing temperature, maybe using adjuvants etc.).

Thank you,