In the Plasmid Mini Extraction kit by Qiagen, they say to elute the DNA with 800 uL of the elution buffer, then to add 560 uL room temperature isopropanol, and finally to centrifuge it for 30 minutes at -4 degrees C. (Then you discard the supernatant, wash the pellet with 70% EtOH, and air-dry).
I would like to increase my yield for obvious reasons. Does adding 560 uL (for a final concentration of ~40% isopropanol) seem low? Should I try increasing it? Also should I try precipitating the DNA at a lower temperature, e.g. -20 degrees C?
Thanks!
Hello Jj,
The procedure you describe is similar to that suggested by the Machery Nagel Plasmid Midi extraction kit (same proportion of isopropanol vs eluted material). I doubt that increasing the isopropanol concentration would increase the yield, as surely the manufacturers of the kits would have recommended it if it worked. A potential problem may be salt precipitation, which may also occur if you lower the temperature to -20 °C. My advice would rather be to take care of not loosing the DNA pellet after centrifugation. I distribute the precipitated material among several 1.5 ml Eppendorf tubes before centrifugation, as it makes it easier to drain the supernatant with a Pipetman without disturbing or catching the pellets. Pellets are then dissolved in a small volume (10 µl in each tube) of T10E1 and then pooled after heating for 5 min at 65 °C.
The problem that will arise with using more isopropanol or chilling the solution is that you are more likely to also precipitate other contaminants. The protocol normally works fine so it is not obvious why you would wish to change it.
- In short adding more isopropanol will not incrase your yield of DNA
- The only thing that really effects recovery is temp; If you chill @-20C for example you might well recover more RNA but with isopropanol you also run the risk of ppt salts: RNA precipitation should always be done at room temp for that reason
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