Can you re amplify the long products with the forward and reverse primers from the original pcrs to selectively amplify the correct length product ?

Thank you for your interest.

Yes I had used the Forward primer of First PCR and reverse primer of the Second PCR and followed the protocol mentioned by the ThermoFischer Phusion Plus DNA Polymerase mannual under 2 step pcr protocol.

Or are you suggesting that I should clean up the Overlap PCR product and again perform PCR on that so as to improve the yield?

The first problem I am facing is not able to detect any colonies after TOPO Ligation, to know what exactly is causing this, I performed gel electrophoresis after loading the Overlap PCR product and extracting it from the gel and found out that after gel extraction I could not detect any bands hence, this might be leading to failure in ligation and thus no colonies seen.

Hope it addresses your question, Thank you.

yes I was suggesting an improvement of yield because at 6ng/ul you will need to use most of your product to get a visible band in an agarose gel and you might still have non specific products invisibly present.

Shreyas Gujar

As I already replied to another question about OE-PCR,

As you know in OE-PCR , overlap region plays role as a primer for each opposite sides to extend the fragment and form a whole segment.

Within my previous experiments, I only got positive result Based on Hilgarth, R. S., & Lanigan, T. M. (2020) method, which is a two step PCR. In first step you need to run a PCR without external primers and only with equal molar mass (same ng) of both fragments you want to attach them together (which you amplified them before separately), for 10-15 cycles and based on overlap region Tm (which is different to external primers Tm). By this step, since you have provided polymerase and other needed materials in the reaction for a PCR (except of primers), overlap region will play role as a primer for each side and form some complete fragments as templates.

Then, in second step, you stop the PCR machine for some seconds and add the external primers (forward of first and reverse of second fragments) and run the PCR for 20-25 cycles, (and now based on these external primers Tm). Now your primers will use formed templates of last step to amplify it in great scale.

So in the gel you must only observe 3 bands, 2 for left and right fragments, and only 1 for attached complete fragment. By good amplification then you need to extract it from gel also by a good concentration (around 50 ng and more/ul) and continue your TOPO cloning.

I also recommend Paul Rutland answer to re amplify your product to increase the yield but since you got 2 bands in the range of interest (2000-3000) which is a bit weird, it would be hard to make sure about target correctness. If you are sure that one of this two bands is your target then re amplificatio to increasing the yield is the best suggestion.

Good luck

The above suggestions are excellent (first run it without external primers or with diluted primers; then amplify with primers). I would add one more point - extent time for extension. 35s are by calculation for 2333 bp, which is very narrow margin. For me, especially with longer fragments, which may be a little more challenging than short pieces, worked best if I extended the extension time to get the Polymerase time to amplify sufficiently. Don't be afraid to go to 45s.

Also, if you have troubles with extraction from gel (like EVERBODY does!), switch to column purification. If you do not have cleaning columns, you can precipitate your DNA with ethanol or isopropanol.

In worst case scenario, you can clone your insert into the vector via PCR by combination of these two papers:

Article Overlap Extension PCR Cloning: A Simple and Reliable Way to ...

Article In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR

where you can split your plasmid into two and create full-length amplicon, which will the bacteria the ligate. If you were interested, I can explain it in more detail.