Hi, 

I am not quite sure about the main cause of this problem, but you can try using mineral oil, this might keep preventing your reaction components from evaporation. 

Good Luck!!

Hemin

You should use mineral oil to reduce the evaporation of the mix.

I think you should buy some higher quality tubes.  This should not happen unless you are using very unusual cycling conditions--really long denaturing times?  Unusally long cycles?

Hi Ahmed,

Nowadays, the PCR machine has advanced to hold PCR tubes without using mineral oil in the tubes. Unless you are using a very old PCR machine. If you are using a newer version of PCR machine, make sure the PCR machine lid is hot (not broken). They are wired to become a heated lid. So, when the PCR cycler is operating, both the top and the bottom of the tubes are hot, making the PCR solution sit tight in the bottom.

Hi Yuan-Yeu Yau,

Probably this is not the case! All PCR machines have a heat-controlled lid, usually at 100 C. But there could be something wrong with the lid, some areas like the first row at which I often notice evaportation. My PCR tubes are of a good quality too.

Hi All,

Thanks to you all Hemin Esmael Othman, Alfredo Procino, John Eimes, Yuan-Yeu Yau, for your suggestions. I will change the tubes and give it a try. 

I think the problem is from thermal cycler... what is type of your cycler machine???? the lid may be not good and its temperature do not reach to 100 C. if the problem not solved, you may use mineral oil to decrease that.

Sounds like a lid malfunction problem.  Try a different, newer machine positive control

Hi Ahmed,

1. NOT all PCR machines have a heat-controlled lid as you stated. The early generation of PCR machines did not have one, so the mineral oil has to be added into the reaction tubes to prevent the evaporation and condensation from the PCR reaction mix.

2. If you want to change the tubes, considering the well-sealed PCR tubes. For that, two things you should look into: (A) tubes have a longer cap flanges (see attached article, page 2); (B) PCR tubes usually are available with domed caps for efficient heat transfer or flat caps for easy piercing and marking (also see attached article, page 2; and attached picture). Consider choosing the tubes with domed caps for well-contacting the heated lid. See more below.

3. See the RG discussion on using domed caps or using flat caps at:

https://www.researchgate.net/post/Does_anyone_know_when_it_is_necessary_to_use_a_dome_or_a_flat_cap_for_PCR_reactions

You may keep the first row tubes empty if your machine is sensitive at that particular area. It happened to me and we did so.  Changing machine might be another option. Good luck 

Hi Asjad Ali and Tiago Oliveira,

Thanks for your suggestions, I will exclude loading samples the first row and change my tubes provider. Let's see whether this solves the problem Thanks much.