How do you mix PCR reactions - swish, flick or nothing? - (Mar/28/2005 )
How do you guys mix your reaction tubes before you place them into the thermal cycler? If your mastermix is mixed well, is it even necessary to mix the tube after you add your template? Just curious what everyone thought.
Small detail I know, but good technique comes from paying attention to the little things.
I heard it's not good to vortex DNA and to pipet U & D enzymes... so flicking looks to me like the best solution.
Simon
Simon
Hi Simon,
I wonder why that is with the vortex? I have been taught to give it a through rodgering and then spin the thing down!
Nick
If I use single 0.5 or 0.2 ml tubes, I may vortex the tubes and then spin down the content. But now most people use strips or plates, vortexing is not so easy especially if you don't have a special centrifuge to spin down after vortexing.
I throw my strips into our super cool eppendorf tube centrifuge that can dub as a plate centrifuge
I've been taught to finger flick and collect the liquid at bottom of the tubes.
The instructions on SYBR green qPCR mixes often say they shouldn't be vortexed.
If you don't have a snazzy plate centrifuge the cheap alternative is either an empty 96 ART tip box or a V-well microtitre plate attached to say 2 ft. long bits of string at each corner. Put your strip tubes in the wells firmly, find an unoccupied part of the lab and swing the plate round by the cords. It takes a little practice, and occasionally you might lose your PCR tubes, but it usually works nicely, as long as there are no members of the safety committee around.
It's also the most fun part of the PCR. How sad am I? 
Hi All
I usually add DNA to tube, then add complete master mix (including Taq) directly into the DNA, this should mix it enough for all reactions as it is a far greater volume than the DNA. I then spin the tubes down, just to make sure.
For the spinning I used a salad spinner, one of those things used for getting water out of lettuce after washing, with a couple of 200 uL tip boxes in it to hold the tubes/strips. It seemed to work pretty well.
Bob
I usually add DNA to tube, then add complete master mix (including Taq) directly into the DNA, this should mix it enough for all reactions as it is a far greater volume than the DNA. I then spin the tubes down, just to make sure.
For the spinning I used a salad spinner, one of those things used for getting water out of lettuce after washing, with a couple of 200 uL tip boxes in it to hold the tubes/strips. It seemed to work pretty well.
Bob
What I do is to prepare a mastermix with everything but the DNA. I flick it energically, spin it, and add it to the bottom of all tubes. Then I store away the PCR reagents and take out the samples (I believe that most contamination comes not from suppossed amplicons floating on the air, but from bad manipulation of the samples). If I had to add mineral oil, I do it at that time. Then I add the samples to the interior part of the cap of every tube, closing it at that moment. Finally, I spin the tubes to bring the samples down and put the tube on the hot thermocycler. This allows me to check that I'm not having pipetting problems with the samples nad also works like a cheap "hot start" PCR by sequestering the sample from the primers until everything is at melting temperature. For me, it works.
Cheers!
i dont mix my mix because i end up with bubbles - surely in a pcr tube there is no need to mix because the volume is so small??? 
Generally i dont mix my final pcr solution...
I mix the template with a small volume of water (~2ul) in a reaction tube. i votex the master mix vigiously before adding taq. after adding taq, i votex a bit (juz a bit). then i put the master mix into the template.
i dont votex or spin down anymore as i guess 94C is hot enough to mix them & the vapour is hot enough to wash anythg on the wall?