27 replies to this topic

[MoleculeMan]

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.

[Simonsays]

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

[methylnick]

Simonsays, on Mar 28 2005, 01:12 PM, said:

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

<{POST_SNAPBACK}>

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

[pcrman]

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.

[methylnick]

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.

<{POST_SNAPBACK}>

I throw my strips into our super cool eppendorf tube centrifuge that can dub as a plate centrifuge  

[5'GCACGTTGGTATAAT]

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?

[bob1]

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

[praveensbio]

5'GCACGTTGGTATAAT, on Mar 29 2005, 10:03 AM, said:

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?

thts nice one....funny but effective enough............

[babylabrat]

Portable microcentrifuge for plates and pcr tubes.

I found a great product which uses a modified salad spinner to spin down plates and 1.5 - 2.0ml tubes.  Check out www.handyfuge.com.  The site also links to a Youtube video which shows you how it works.  
I think this would be great to use in the field since it is essentially a portable centrifuge.

[jiajia1987]

I always flick and spin down the contents

[alan6017518]

I vortex it and spin it down when its in separate tubes, as for strip tubes I haven't vortex them, may be I can give it a try with the handyfuge, that thing looks funny.

[Kami23]

alan6017518, on May 28 2009, 04:14 PM, said:

I vortex it and spin it down when its in separate tubes, as for strip tubes I haven't vortex them, may be I can give it a try with the handyfuge, that thing looks funny.

You dont need anything fancy! we use a salad spinner too... we call it saladfuge!

[biotechnica]

All work, even not mixing! Things mix when the liquid gets hot and evaporates and comes down (heated lid cyclers).

[jiajia1987]

Always just tap gently at the base of the PCR tubes and spin down the contents for 10s.  Things sometimes get a little weird without mixing.

[Atul Kumar Gupta]

jiajia1987, on Jun 23 2009, 07:19 AM, said:

Always just tap gently at the base of the PCR tubes and spin down the contents for 10s.  Things sometimes get a little weird without mixing.

By the taping i am found air bubble in the PCR tube, this air bubble create problem in analysis of QPCR. so please suggest procedure for mixing the master mix or aliqute in the PCR tube.