I recently extracted DNA from iPS cells using cell lysis buffer + prot K followed by ethanol precipitation. The resulting genomic DNA is viscous and hard to pipette. Any thoughts on how to reduce the viscosity? Should I treat with additional prot K or RNase? Should I resuspend in a greater volume?
I am a rheologist, so I will provide some thoughts from a rheology viewpoint
The viscosity of DNA solution scales with viscosity exponentially, with an exponent of about 3.4. This means if you increase the volume by a factor of 2, the viscosity will decrease by a factor of 2^3.4 ~ 10. The viscosity also depends on molecular weight in a similar way.
Increasing the temperature will NOT help too much, since it is way above the glass transition temperature Tg.
Hope this helps.
I am a rheologist, so I will provide some thoughts from a rheology viewpoint
The viscosity of DNA solution scales with viscosity exponentially, with an exponent of about 3.4. This means if you increase the volume by a factor of 2, the viscosity will decrease by a factor of 2^3.4 ~ 10. The viscosity also depends on molecular weight in a similar way.
Increasing the temperature will NOT help too much, since it is way above the glass transition temperature Tg.
Hope this helps.
Do you need your DNA unfragmented? If your using if for PCR, southern blot and others you can use a syringe and pass your sample through an insulin neddle and the viscosity will be reduced (as Yanfei said it is a function of Mw and by this method you're reducing it).
Reduce the number of cells you are starting with. I am not sure if using more volume of lysis buffer/ethanol will help. Ethanol helps precipitate the DNA thus "coming out of" the solution. I therefore think what I would do is use fewer cells to start off
Viscosity of DNA depends drastically on the solution ionic strength. More salt - less viscosity.
The easiest way is to add more TE buffer or water and mix well using a pipette or vortex gently. If resuspension is achieved, may also be heated to 56 degrees Celsius.
The viscosity of DNA solution decreases after treating by ultrasound.
There are several ways to solve your problem. But which one accept depends on the goal of your work.
Thanks for all your responses. For my purpose I need unfragmented DNA. We start with one ~80% confluent well from a 96-well plate. iPS cells are relatively small cells, so I would guess the cell number is on the high end for a 96 well plate. Our goal with the genomic DNA is to test for integration using PCR.
1)For PCR you don't need whole DNA, but integration locus and primers for the site.
2) As I wrote, you can decrease viscosity of the very high molecular weight DNA adding salt up to 1 M or alkali (pH 13.2) to denature DNA.
Good luck.
I've has similar issues with some samples of genomic bacterial DNA. Have you made sure that your DNA isn't contaminated with sugars or proteins? If the result of a DNA extraction is abnormally viscous, I look for unexpected peaks using a Nanodrop; another sign of DNA contamination is difficulty loading in a gel.
Dissolve in a more volume of 0.5-3 M NaCL TE buffer and heat briefly at 56-63C. You can also use a syringe and a vortex.
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