How do you mix PCR reactions - swish, flick or nothing? - (Mar/28/2005 )
I have never vortexed after adding to individual tubes because high temperature is enough to cause thermal agitation mixing so I never feel need to vortex.
I loved reading the answers to this thread ... home-made centrifuging devices, well-funded labs with good toys and then people asking "why do you mix at all?"
I do like Bob does ... add my template to the tubes/strips/plates and then pipette the master mix over the top. No mixing then.
But I DO briefly vortex the master mix (I now use a supermix and just add primers) and then quick spin to get the liquid out of the lid.
In the olden days, the theory was that you should never vortex enzymes since it was thought that would shear them. Having worked with restriction enzymes, polymerases etc for 15 years, I have never lost activity by quickly vortexing a master mix.
What other theories/ stories are "out there"??? (I love a good laugh )
Hi gang. First reply as a new memeber.
When doing either single-tube pcr or 96 well plate pcr, I make up my mastermix and set it on ice. Then add the template to the tubes. Then I add the master mix and pipette up and down 4 or 5 times to mix. No bubbles, no vortexing, no spinning.
This same philophy extends to restriction digests.
Small detail I know, but good technique comes from paying attention to the little things.
to me it's all dependent on what type of pcr im doing. With QPCR, eliminating tube to tube variability is of the most importance especially when running a standard curve set. So I do a real quick pulse vortex to make sure my master mix is mixed well and then centrifuge down.
For just about everything else, a couple of flicks and a whip will do.
I agree with some of the others at the bottom of this page, you definately need to mix your master mix (very brief vortex and equally brief spin-down) but I add this to templates already in the tube, and I believe that the denaturing cycle will mix the template and PCR reagents sufficiently. The only case where I do any centrifugation or mixing (even with a pipette) is if there is an air bubble seperating the master mix from the template.
BTW--in regard to vortexing enzymes, we always mix our restriction digests either by pipetting or a brief vortex. My boss has a story from grad school where a guy could never get his digests to work--turned out the enzyme/glycerol sank to the bottom of the tube and b/c he did not mix it wouldn't cut the DNA....
I vortex then whip down to the bottom. Using a heated lid means it's not really necessary to spin down as the liquid will collect at the bottom of the tube anyway
i have always learnt that vortexing DNA is a No No... so i either finger flick, or tap in on the bench, or most commonly pipette swish!
i would not vortex Genomic DNA if i wanna do fingerprinting, lesser fragments would be better.
if i am trying to amplify a specific region vortexing is all fine
for small templates like plasmid or restriction fragments vortexing is fine
of course i will tap the multiwell plates if i am making many samples
Thank you! Never thought that something so simple could turn out to be so interesting! I always vortex and spin everything, smples, reagents, mixes... except when I'm advised not to do it (like with DNAse l). I liked the sugestion of Bob, it makes sense and I will try it.
I've made a little experiment about the effects of vortexing in the integrity of DNA, using diferent methods of extraction and diferent vortexing periods of time. I've tested the DNA using a multiplex pcr for diferent fragments sizes, and vortexing did not affect DNA integrity. You can vortex all you want!
Sometimes I vortex and spin, sometimes I flick, sometimes I pipet, and I've never seen any difference. Though I guess that vigorous vortexing or pipetting can shear large DNA templates (genomic). For the mixing purposes, the PCR tube will be heated where the walls of the tube connect with the cycler, this will create a slight difference in the temperature in the liquid at the walls and in the center of the tube. Although these differences will be small in a thin-walled PCR tube, it should be enough to create convection streams in the liquid ensuring proper mixing.