
Vortexing genomic DNA, detrimental or not?
#1
Posted 07 May 2009 - 04:18 AM
What do you do? Are vortexing OK or just a plain no-no?
#2
Posted 07 May 2009 - 05:54 AM
I read somewhere that vortexing genomic DNA solutions is a definite no-no (because of strand breakage etc?). However, it may somehow look like our purified DNA aggregate to some extent upon thawing -three parallel measurements can give as much as 2x concentration difference. if we vortex our DNA samples the three parallel measurements are all identical. We use the nanodrop so I can't tell whether the vortexed DNA contains any broken strands or not but at least it looks like we have to shake the samples thoroughly after thawing.
What do you do? Are vortexing OK or just a plain no-no?
Hi,
Vortexing genomic DNA is not a good idea, it leads to breakage of DNA strands. For that matter, any sort of mechanical pressure (like vigorous mixing etc.) increases the chances of your ending up with sheared genomic DNA. I keep my gDNA solution at 4o, so that there is no problem of thawing everytime I need it.
#3
Posted 07 May 2009 - 06:40 AM
Albert Einstein
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie
#4
Posted 07 May 2009 - 07:17 AM
#5
Posted 07 May 2009 - 07:27 AM
#6
Posted 07 May 2009 - 08:13 AM
I have another genomic DNA question. When doing PCR, i use 10 ng of plasmid DNA for my template, do you have to use more genomic DNA then that to amplify a gene?
Generally yes.
But sometimes more is not better. Genomic DNA can sometimes be contaminated with polymerase inhibitors which despite your best effort can not be removed. In such situations, diluting the genomic DNA sample with water and using the diluted DNA (which has diluted inhibitor) will actually yield a better PCR signal
#7
Posted 08 May 2009 - 12:49 PM
#8
Posted 10 May 2009 - 11:15 PM
You might want to consider diluting the gDNA; that way your spec readings may become consistent.
#9
Posted 13 May 2009 - 08:58 AM
Cheers