I wouldn't recommend vortexing SYBR Green, either before or after making the master mix. I briefly vortex and spin down the primer of interest, mix the total master-mix (with sybr) by inversion, and do a quick spin down (a pulse or so). Vortexing after adding Sybr poses the risk of damaging the Taq pol.

Let me clear for why you want to do qpcr and for what purpose.

If you want to drawn linear graph and absolute quantification then you have to to serial dilution. For this first you should define copy number.

If u want do some diagnosis or some relative expression then no need to do serial dilution.

I prefer to vertex master mix once it is prepared. The master mix is always without templet and spin down.

After adding templet to PCR tube/strip/plate the mix by tapping and spin down and make sure that their should me no air bubble.

If u mix by pipetting the chances of getting air bubble is more.

Brief vortexing (2sec) is recommended for master mix mixing followed by a brief spin down. Primers dilution can withstand vortexing, once enzyme is added pipetting mixing is best followed by brief low speed spin down. cDNA is more stable than mRNA and can withstand short vortexing but for dilution i always do pipette mixing before serial dilution or do pipette mixing of dilution and a brief 4,000rpm spin down before taking an aliquot. In general enzymes stored at lower than 4 degrees celsius and RNA should not be vortexed. Hope this helps

Vortexing intensely can cause enzyme inactivation as well as bubble formation in the mixture. Usually, we leave each solution to the wall of the PCR tube and mix the PCR mixture by pipetting and thus preventing bubble formation. Finally, spin the tubes for few seconds.

Mix your sybr green, primers and DNA individually in their tubes. Then take a separate tube and add cDNA, then primers and nuclease free water and lastly sybr green. Immediately centrifuge for 5 sec and run the reaction.

Hi Syed Monzur Morshed ,

I agree with the previous comments.

I usually mix by vortexing cDNA+primer dilution. After that adding SYBRGreen Mix mixing by pipetting. If you vortex strongly the whole mix with SYBRGreen, It could damage the Taq pol.

Previous adding the mix to the plate, I spin down or gently vortexing and centrifuge it (only one pulse.... you know)

If You have bubbles in your wells, after loading the plate, You can use a plate centrifuge for removing it.

Hope this helps!

Best!

Gentle vortex is ok

I wouldn't recommend vortexing SYBR Green, either before or after making the master mix. I briefly vortex and spin down the primer of interest, mix the total master-mix (with sybr) by inversion, and do a quick spin down (a pulse or so). Vortexing after adding Sybr poses the risk of damaging the Taq pol.

Hi Syed Monzur Morshed , in general, vortexing primer before adding into the master mix is a good idea. I prefer to vortex the cDNA also before I add those in the qPCR plate. I have mixed opinion on vortexing the master mix that contains enzyme and the answer is it depends on the commercial kit. I have experience with two kits, one from Agilent and another one from ThermoFisher (Power SyBr). The Agilent one didn't work well after vortexing (huge variations in my technical replicates). On the other hand, ThermoFisher worked well after vortexing (technical replicates didn't have much variation).

i have not any idea.

Enzymes should be spin down always and should not be vortexed..