Scaling up PCR reactions - (Apr/05/2007 )
Quick question. Is there any reason why scaling up of a PCR reaction, say 15 uL to 30 uL or 25 uL to 50 uL would fail? I had great sucess running my samples in a 15 uL pcr reaction for testing purposes, however due to the need for a large amount of product for sequencing I scaled the reaction up, doubling all components to a 30 uL reaction and rather than simply doubling my yield i seemed to get less product in a number of my samples. I'm not sure why. Master mix was of pretty standard conditions 200 nM of each primer, 200 uM dNTPS, 1.25 u Taq.
Not sure whats going on. Any help would be appreciated.
Are you changing the settings on your cycler so that it knows the volume of the PCR reaction? The thermal mass of the liquid can change the speed of cycling. This is especially important with fast cycle conditions, such as 10 second denature and anneal cycles.
Have you changed the setting on the thermocycler from 15ul to 30ul? Make sure the machine knows that the volume within the tube is 30ul rather then 15ul.
As for thermal cycling, I think most machines do fine till about 40 - 50 ul. After that the lag between temperature cycling becomes quite significant and thus inteferes with the PCR reaction
Set up 5-6x reaction tubes of the 15ul reaction and run PCR. you should have more.
if you plan to change volumes, you need to adjust settings in the machine.
You get a better yield with lower volume reactions, for some unknown reason. I think Tavi investigated it but never found out the reason.
When I first started doing PCR I did it in 20ul volumes because people told me "there's no point doing anything larger". Now I do 10ul, for exactly the same reason!