2 replies to this topic

[cyphlurm]

Dear BioForum'ers,

I have to extract DNA from a small number of FACS-sorted cells (around 10.000 to 1000 cells pr. sample). I've tried with a normal column-kit from Qiagen, but after about 100.000 cells, I loose to much DNA in the membrane.

Do you have suggestions on what other solutions I could try?

Edited by cyphlurm, 30 July 2012 - 11:21 AM.

[Trof]

I was dealing with such problem. I figured out that main problem on the column kit is the loss on the column and high elution volume, so I tried Trizol.
It's exactly not very suitable for isolation of DNA, since DNA from it is disolved with difficulty, but it omits the column. I used standard protocol for 500 ul of Trizol (you may even try lesser amount but in smaller tube) and acrylamid carrier for helping precipitation and pelet visibility. Otherwise carried out as in the protocol for DNA isolation. It's good you can dissolve in any amount you want and thus to concentrate the DNA. I don't use the NaOH as they recomend for redissolving but heat it to 50 C instead for 10 minutes. Still, I had small colonies varying in size around 100-1000 cells and didn't get positive signal on genotyping every time.

Depends what you need your DNA for, what purity and aplication, when I asked about other commercial solutions, some company representative suggested to just boil the colonies for 10 minutes in small volume to deaturate proteins and put it right into PCR. Didn't try that though.

[cyphlurm]

Thanks for the suggestions, I will try it out tomorrow if we have all the stuff in lab - I will let people know how it goes :-)