8 replies to this topic

[DocFlow]

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?

[Vini]

DocFlow, on May 7 2009, 05:18 AM, said:

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 4^o, so that there is no problem of thawing everytime I need it.

[merlav]

To mix the DNA/RNA/oligo is better to flick the tube

[perneseblue]

and to be really careful with gDNA (>150 kb), use plastic pipettes with their tips cut off and not standard pipette tips

[shimshady]

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?

[perneseblue]

shimshady, on May 7 2009, 07:27 AM, said:

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

[phage434]

The handling of the genomic DNA depends on the goal of the isolation.  For PCR, there is no problem with vortexing.  For cloning BACs or cosmids, you may need care.  For pulsed field gel, even careful handling is not sufficient, and in-place digeston of cells encapsulated in agarose is required.

[swanny]

Do the experiment! Run a gel of your DNA prepared in different ways; see what differences in size you generate.

You might want to consider diluting the gDNA; that way your spec readings may become consistent.

[Roberto]

I recommend to store genomic DNA at 4°C over a drop of cloroform to avoid an eventual contamination.  It can then digested without apparent problems.
Cheers