4 replies to this topic

[muntasir]

I am doing lots of real time PCR nowadays. It is about 4/5 plates per day. I was planning to prepare all the master mix together, dispense into 96 well PCR plates, add template and keep the plates at 4^oC to run one after another. Usually, one run takes about 1.5 hours. My supervisor suggested me to reaction mix just immediately before running. He is afraid that it could hamper the reaction. To satisfy him, I need some references which suggest “We can prepare PCR reaction mix prior 3-4 hours of run.

Can anyone suggest from his/her experience anything? I am looking for articles. Please provide me some information if available.
I am doing lots of real time PCR nowadays. It is about 4/5 plates per day. I was planning to prepare all the master mix together, dispense into 96 well PCR plates, add template and keep the plates at 4^oC to run one after another. Usually, one run takes about 1.5 hours. My supervisor suggested me to reaction mix just immediately before running. He is afraid that it could hamper the reaction. To satisfy him, I need some references which suggest “We can prepare PCR reaction mix prior 3-4 hours of run.

Can anyone suggest from his/her experience anything? I am looking for articles. Please provide me some information if available.

Note:

Our machine: ABI 7500
Reaction Volume: 25 microlitters
Organism to be detected: bacteria, virus, protozoa
Minimum Time we want to keep our master mix in 4C: 1 hour 40 mins.

[leelee]

I think it should be fine the approx 2 hours you've said. We do it sometimes, with no apparent reduction in sensitivity or any other issues. In fact, once thawed, I keep my qPCR master mix at 4C (the repeated freeze thaw is much more of a concern). I'm assuming your enzyme is a hot-start too, so it shouldn't be doing anything funky before the reaction is started with the initial high temp melt.

But then I'm no expert so hopefully someone can give you more than just a "I think its ok"

[Trof]

Depends on the master mix.
Roche quarantee final PCR mixes to be stable 24 hours at room temperature.
Check with your mix suplier.

[Ameya P]

Not of great help, but I usually set up my usual PCR reactions, one after the other so that I am free to do other things (like Bioforum )for the remaining part of the day.

I set up my reactions on ice and once done, I return the ice pack along with my tubes to the freezer. I remove my tubes and put them on the thermal cycler when I can during the day.

[muntasir]

Thank you all for your advice. I also think it would not influence the result if I keep the mastermix for 2 hours in refrigerator. but it is safe to have published reference. Any contribution is welcome..