Hi Fernanda,

I have never cloned a RT- qPCR Product directly. However, I think that this could work as the amplification is done with a Taq-Polymerase. However, I would first check on gel how your PCR product look like and if you have on nice distinct band. If this is the case I would go ahead. Otherwise consider conventional PCR. I did a lot of cloning with TOPO-TA and usually this works very well. I ususally did the cloning without cleaning up the PCR product - but you can clean up the PCR product so that you would get rid of the other components of your reaction. For cloning I often used less than 1 µl of the vector (usually 0.5 µl is sufficient). Don't use to much of your PCR product - if you have a strong clear band when you put 5-10 µl PCR product on the gel 1 µl PCR product should be ok. For the rest follow the manual - a blue-white screening is recommended and I would do it if possible, however if it works most of your clones should be positive. And I hope you have also ordered the competent cells as this is much more efficient than self-made competent cells. Another tip - do not wait to long with the cloning reaction after the PCR because you will start to loose the A-overhangs. Leave the PCR-product on ice during the time the gel is running.

I want to add something to my previous answer, because I didn't take this in consideration. If you do a normal PCR you usually have a final extension step (72°C , 5-10 min) to fill up the ends of PCR products. Usually you don`t have this step during real-time PCR. So I am not totally sure if you can get problems here with incomplete sequences at the ends (and in the end incomplete insert sequences in your vector). You can still try, however for cloning it might be easier to created cDNA from your RNA and do normal PCR. As the cloning process is fast with TOPO-TA this can be done in one day.

Good luck

Thank you, Dörthe Andrea Kesper ! Maybe I'll try cloning the same sample both ways and compare the efficacy. Your comments were very helpful to guide the next attempts.

I'll update you how it went!

I am interested to know how it will work - but I am optimistic :-)

Yes