Dear Marusa,

            If you prepared the mastermix without enzyme you can store it at -20 degree. All the best.

Hi, thank you for the answer! Unfortunately I did add the enzyme. Does that mean it is ruined and I have to make a new one? At least for the one with the enzyme, the NoRT control doesn't have the enzyme anyway...

Hi Marusa, 

Prem is correct. If you are interested, here is some further information regarding your question:

The problem with freezing your mastermix with the enzyme already added is that freezing certain active enzymes such as the polymerase family of enzymes tends to result in diminished or ablated enzyme activity - in other words, freezing your RT enzyme will likely 'kill' it. This is likely true for all enzymes. Why this is the case, I'm not entirely sure - it may have to do with the formation of ice crystals around the enzyme leading to permanent reconfiguration of the tertiary structure of the enzyme (which is of course required for its activity) or even damage of the polypeptide chain.

You may notice that most enzymes you purchase or come across in lab are stored in a viscous solution. This is generally a solution containing around 50% glycerol. Glycerol is widely used as a relatively inert chemical for storing things such as active enzymes and even bacteria (more on that in a moment) because it has a very low freezing temperature. Your mastermix wouldn't contain sufficient glycerol, and would freeze, 'killing' the enzyme.

You may also be familiar with GoTaq green mastermix - which is generally frozen upon arrival. This solution contains everything necessary for PCR including the polymerase enzyme, excluding primers and extra water. While the reaction mix is proprietary (its a company secret), its well known that while PCRs using this mix do indeed work well enough for most things, the efficiency is far lower than higher quality Taq reagents... namely those you would find stored separately in glycerol.

For a sensitive reaction like an RT, where you need maximum efficiency so that you don't miss the conversion of ANY RNA to DNA, then you need to ensure that your RT enzyme is working at max capacity.

Finally - I regularly clone sequences of DNA to use as in situ hybridization probe templates. When we successful get the cloned sequence in to a plasmid vector, and successfully grow bacteria that have been transformed to carry that plasmid, we mix 500uL of the bacteria (in Liquid Broth) with 500uL of glycerol and create a bacteria stock. We then place this tube at -80C for long term storage. Any time we want to grow more bacteria, all you do is scrape off a little ice from the top in to a tube containing LB. 

I've attached a couple links related to this post for reference or verification if you're keen. I hope you've found this information useful.

All the best and good luck on the science.  

http://www.biotechniques.com/multimedia/archive/00011/00293st06_11772a.pdf

Thank you so much for all this information, Paul! It was indeed helpful.

I will prepare a fresh master mix and learn from my mistake :)

Good explanations Paul

Very useful info Paul!