4 replies to this topic

[JustCurious]

hello,
Im doing DNA extraction for MCF-7 and HeLa cells. They have undergo drug treatment and I just want to look at their DNA fragmentation (if any). The problem is, when I wash them with EtOH, centrifuge, dry the EtOH, i suppose to have a pellet or perhaps some debris on my tube. but instead of that, i have a bulk of gooey sticky stuff.. i discover that the gooey sticky stuff is the DNA because when i run gel, they all have bands. but unfortunately, with some RNA below.

i hope to know is there any possibility to reduce the viscosity of that gooey stuff and how can i eliminate the protein content in that gooey stuff? i have read alot of discussion here. but most of them is about how to eliminate the DNA and getting the protein.

Edited by JustCurious, 06 October 2012 - 07:34 PM.

[bob1]

The typical way to do a DNA extraction is to use solutions that will dissociate/denature/degrade the proteins such as a proteinaseK digest followed by phenol:chloroform extraction, then precipitation of the DNA.

You can also use DNA extraction kits, which are relatively expensive, but fairly quick.

[JustCurious]

thank you for your advice.

i had try use the PCI method but the gooey thing still exist. I wonder if I can increase my Proteinase K concentration or increase its incubation time. right now, my Proteinase K concentration is 400ug/mL for about 1.5 x 10^6 cells and i incubate them for 30 min at 65C.

Only if my conventional DNA extraction is really not working, then only my supervisor give me kit. right now, Im trying my best to use the conventional one

[bob1]

You can certainly increase the proteinase K time.  I used to extract DNA from tail tips using an overnight incubation at 37 in proteinase K.  I wouldn't increase the concentration though, this will only increase the amount of protein that is being carried over into the extraction.

You could try lowering the number of cells you are trying to get DNA out of - it may be that you are overloading the capability of the system.

[JustCurious]

i guess so. i had too many cells in my 6 well plate. after 3 days, some of them even floating in the medium. well, thank you again