Hello,
Today I want to ask about handling of cDNA during RT-PCR.
Whatever i do, i think those seem to be trival occasionally affect research
results quite critically.
I always voltex mixture with many components.
And I conduct tapping before using
reagents to resuspend components
sunk in the bottoms.
When it comes to cDNA, I wonder whether cDNAs are evenly distributed
in specific sample(1.5ml tube) even without tapping or voltexing.
My cDNA samples are very low in volume(Just 20ul) so I think cDNAs are
evenly distributed without sinking problem.
Like all researchers, I am very busy with many works so I often
cannot remember in detail whether I voltexed or tapped my cDNA samples.
I always try my best to prevent any mistakes in my works with my concentration.
And if cDNAs are evenly distributed in my specific sample without any tasks, are they still evenly distributed after voltexing, inverting, or tapping, etc?
As mentioned above, my cDNA sample seems to be very low in volume and height.
Thank you for reading!!
Whatever the solution, whether cDNA,RNA ,DNA or beads etc. always do gentle pipetting 5-10 times (ups and down) with a retention free tips to use, rather vortexing or tapping, because that is quite harsh treatment.
Vortexing can shear cDNAs. You can finger flick the tube and quick centrifuge or pipette up and down a few times.
I would not vortex DNA , Firstly it may break the long , thin strands of dna....gentle mixing is much better. Also vortexing often creates bubbles and the concentration of proteins and dna in bubbles is different from that in the original solution ( higher ) .It is a good idea to gently spin reagents down to the bottom of the tube to ensure that all of the reagents are in the reaction mixture and then I would gently mix by flicking a few times and after that thermal diffusion will keep the reagents mixing. For small volume reactions do not use 1.5ml tubes as the volume above the reaction mixture is large and you will get evaporation of the water and the concentrations of all of the reagents will change.Sometimes you will even get condensation on the lid of the tube in long reactions. Using small tubes is better and either a pcr machine with heated lid or a water bath is best rather than a hot block or pcr machine without the heated lid to minimise evaporation with small volumes
Like all the answers above, I would suggest not to vortex but just gently pipette your sample up and down a few times or flick the bottom of the tubes and a quick spin down using a mini centrifuge
Don't vortex nucleic acids. As mentioned above either finger flick your tube followed by a quick pulse or gently pipette several times.
Adam and Zimm in 1977 reported that in solutions used E. coli DNA was reduced to half molecules at shear stress of 0.4 dynes/cm 2, whereas De la Paz et al. in 2012 found that a shear stress of 10 dynes/cm 2 may be produced at the rotating frequency of 195 rpm within the cell culture medium system...
Dear all all the answerers,
Thank you for your replies!
Great help for me!
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