27 replies to this topic

[volano]

I prefer to spin down, sometimes drops would adhere by the tubal wall.spin the tube for 3-5 sec is ok~

[massiveattack]

Hi,

From my understanding it's not advisable to vortex genomic DNA as it is more likely to nick.  Plasmid DNA is more hardier and can be vortexed (e.g. vortexing when doing minipreps).  With regards to PCR, I make a master mix (containing polymerase if I'm doing a pseudohot start PCR) which I vortex to mix, then aliquot it out into PCR tubes.  I add the DNA with a multipipette and the force of release mixes it in itself without the need for flicking or pipetting up and down.  If I'm doing a hot start PCR, then the polymerase is added only after master mix and DNA have been denatured at 95oC for 2 minutes.

Cheers,
massiveattack

[chromatin]

MoleculeMan, on Mar 28 2005, 02:08 PM, said:

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.

If master mix is mixed well, I pipet DNA template, and do not bother to mix it. Never had any problem.


http://www.bioprotocols.info

[liugang]

I always flick and whip then spin 5S,it is OK

[cbernst1]

Here's some more detailed help with mixing PCR reactions...  



http://www.scientistsolutions.com/PrintTopic.aspx?id=ee7eb842-51a2-4d24-bcfd-3d020058d1ee

[DNAgeek]

I flick, but gently enough so that I don't have to spin. (Try it with a nice thick bromophenol blue-in-glycerol loading dye instead of a sample and you'll see what is effective). That being said, when I run real-time quantitative PCR, I add the sample to the buffer that I already put in the tube and just rinse the pipette by pipetting up and down. Real-time PCR is very sensitive and this method works well, so I think it should be more than sufficient for regular PCR.

[Biotechwoman]

My Teacher always tells us to never vortex when working with DNA (only exceptions is breaking down yeast for and extraction) especially when you are doing PCR. Vortexing will shear your DNA and then you will have different sized pieces being replicated.

[Ikar]

I prefer stripes with 8 reaction tubes. So to accelerate the whole thing (I don't want to have the polymerase too long in my reaction tubes before they enter the PCR cycler)
I just mix them by flicking and then briefly (5sec) centrifuge everything down.

[sxh999]

I always flick the tubes and then spin the solution down. The detail is I usually don't spin them hard, just turn on and turn off immediately. Hope this helps. Holly

[Laforet]

We add up to 10% DMSO for GC-rich fragments and the only way to mix it properly is vortex and spin

Strip tube rotors are essential

[scolix]

Flick the tube and spin it down.

[4N6Addict]

methylnick, on 28 March 2005 - 06:07 PM, said:

pcrman, on Mar 28 2005, 05:56 PM, said:

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  

Exactly what I do!

[4N6Addict]

chromatin, on 04 July 2010 - 08:38 AM, said:

MoleculeMan, on Mar 28 2005, 02:08 PM, said:

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.

If master mix is mixed well, I pipet DNA template, and do not bother to mix it. Never had any problem.

I always put my DNA in the tubes before I add my master mix so that the mix doesn't have to sit as long before I use it, so pipetting the DNA isn't an option for me.  Does sound like the best option if you're going to add the DNA last though.