3 replies to this topic

[lotus]

Hello,
Some people seem averse to doing overnight 4 C holds  for their PCr reactions, while some don't mind. Does this really affect the PCR machine? If so, is it better to hold at a higher temperature like 10 C.

[bob1]

Holding at 4degrees can cause water vapour to condense on any part of the block that is exposed to air.  If it condenses on the underside it may drip into the electrical components causing malfunction.

It is not necessary to hold your products at 4 deg or even 10, DNA is very stable and will last at room temperature for quite some time.  The reaon people hold at a lower temperature is to limit the activity of Taq or other polymerase so as to prevent potential non-specific binding of primers and amplification from these sites.  However, unless you are planning on leaving your stuff at room temp for several days, this will not be a problem, as most PCRs of 30 steps are limited by primer or dNTP availabilty.

[mdfenko]

one other thing. many thermal cyclers use the peltier effect for heating and cooling. this relies on the movement of ions to or from the block. extended holds at 4C will cause a more rapid depletion than with a 10C hold (or no depletion at room temp).

it's a good thing that the blocks are replaceable in most cyclers.

[jiajia1987]

The laboratory I am working in keeps holds the PCR products at 16deg as they said it can cause the PCR machine to spoil because of the condensation. Personally, I am more comfortable with keeping my products at 4deg but so far, keeping it at 16deg has no problems for me.