I am performing phenol-chloroform-isoamyl alcohol DNA extraction method for cattle semen samples. I wash the pellet with 70% ethanol, air-dry/dry in incubator at 37deg and dissolve the pellet in TE.
My A260/A280 ratio is fine.
However, for many samples my A260/A230 is between 0.5 to 2. How do I improve this ratio ?
Thank you in advance for your replies!
Many contaminants can give you a low 260/230 ratio, the most obvious is phenol. To remove phenol contamination from your samples you can wash twice with chloroform prior to precipitation and washing with 70% ethanol.
Do you use Proteinse K during your DNA isolation? It might have to do with to much protein in the sample
Bonnie Molenaar Yes I added proteinase K.
@Daniel Martin I need to add chloroform to the aqueous phase, centrifuge it, collect the aqueous phase and repeat the process again ? Can you clarify this?
@Arshed Kadim: Yes, I will use nanodrop.
Thank you all for suggestions !
If you already added proteinase K than after washing with 70% ethanol, you may wash it once with absolute ethanol, and then add TE buffer in it after drying.
What I do for DNA extraction from spermatozoids.
I send you this protocol in attachment document
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