When elute my plasmid from the column, I get a great amount of plasmid DNA in the elution buffer (~192 ug DNA in 5 ml). After the precipitation with isopropanol (3.5ml) and wash with 70% EtOH (1.5 ml) I clearly see the pellett. When I redissolve my plasmid in H2o I get 1,6 ug DNA in 40 ul.
What could cause DNA loss during precipitation and washing?
Isopropanol is more suitable for high molecular DNA (chromosomal or plasmid), and done in ambiant temperature. Since isopropanol is less volatile than ethanol , and is not effective to eliminate trace of organic compounds, So try to wash twice with Et-OH.
Do you add salt in your precipitation ? if not you have to add Na-acetate 0.3M.
Do you wash with ice-cold Et-OH?
Hope it helps
Isopropanol is preferred for high molecular weight DNA, (chromosomal or plasmid), The precipitation is faster and can be done at room temperature. In contrast Isopropanol is less volatil than Et-OH and is less efficient for eliminating organic compounds. for this reason you should wash twice with ET-OH.
In your precipitation, You must add salt for best DNA recovery, 0.3M of Na-acetate must be added ib both isopropanol or ethanol precipitation. I think this is the main reason why you have a DNA loss.
the lack of salt addition could be one of the reasons since I did not use Na acetate. So you suggest that after elution precipitate and wash with ETOH? When should I add the Na acetate?
thank you
Add Na-acetate to your DNA, Add isopropanol for precipitation. the final concentration of the salt must be 0.3 M.
Centrifuge, and wash with Et-OH.
One more disadvantage of isopropanol precipitation is that DNA precipitated by isopropanol does not stick tightly to the wall of the microcentrifuge tube and is easily lost.
If you have this problem, simply switsh to the ethanol precipitation protocole.
Good luck
According to manufacturers' instructions, if you use a column-based kit (e.g. Qiagen Midi), there is no need to add salt to the eluate prior to isopropanol precipitation. My guess is that you lost the pellet while discarding the supernatants of the isopropanol precipitation or of the EtOH wash. Did you still see your pellet after discarding the EtOH wash ? My experience is that the supernatants have to be removed very gently with a pipetting device, while carefully watching that the pellet doesn't come off and get sucked along with the supernatant.
Thanks for the answer!
There was a stupid mistake by me. After the precipitations, I clearly saw the pellet in each case. The EtOH was not perfect.
Today I got 2,5 ug/ul cc, so now it works! Thanks again.
Hi,
You may also use carrier molecules for efficient precipitation such as LPA, tRNA or Glycogen
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