I am looking for a simple but effective method to extract DNA from cells after drug treatment in a 96-well plate set up. The amount of cells are low (in the range of 10,000-30,000 cells) so any method that would maximize the amount of DNA extraction, with less interference with PCR is what I am looking for. I have tried various kits (chargeSwitch, magnetic beads) but since these kits involve wash steps, I have not had consistent success with these methods.
There is another factor that the media/drugs coudl interefere with the PCR, so I will need to have atleast one initial wash steps to remove the media and resuspend the cells. Instead of kits, should I just try heating method to lyse the cells and run PCR? Any thoughts on how this works?
I am looking for any help or suggestions regarding this.
Thanks in advance!
Assuming your cells are eurkayotic and not bacteria, heating alone will not work, you need to digest the histones away.
There are 96 well DNA purification kits from all suppliers (sigma, qiagen, lifetech...). If your PCR is simple, you could do a manual DNA extraction (lysis buffer, proteinase K treat, ethanol precipitate) all in the 96 well plate. This will work out very cheap, whereas the 96-well kits cost a fortune if you have many plates to get through.
This protocol looks good to me. http://mcmanuslab.ucsf.edu/protocol/dna-isolation-es-cells-96-well-plate
as does this:
http://www.cgm.northwestern.edu/docs/ttml/Isolation_DNA_96_Well_Plate.pdf
I suggest manual (phenol) extraction such as with Trizol (Life Technologies). This gives you more control over quantity and quality of DNA and RNA you extract, depending on how careful you are with pipetting at each step involved. This may be ideal for the 10,000+ cell number range you are working with. Although time consuming, but I have had very good yields of RNA and DNA with the reagent..
Assuming your cells are eurkayotic and not bacteria, heating alone will not work, you need to digest the histones away.
There are 96 well DNA purification kits from all suppliers (sigma, qiagen, lifetech...). If your PCR is simple, you could do a manual DNA extraction (lysis buffer, proteinase K treat, ethanol precipitate) all in the 96 well plate. This will work out very cheap, whereas the 96-well kits cost a fortune if you have many plates to get through.
This protocol looks good to me. http://mcmanuslab.ucsf.edu/protocol/dna-isolation-es-cells-96-well-plate
as does this:
http://www.cgm.northwestern.edu/docs/ttml/Isolation_DNA_96_Well_Plate.pdf
Thank you very much for the replies! I will try the direct extraction method and see how it works in my case. Thanks for the protocols Nathan!
Just wondering, since the cells would have been inubated in drugs (different concentratin) will the drug have an effect on the PCR reaction? Ofcourse I will first spin down and wash off the supernant, but just wondering if this woudl be enough?
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