We use phenol-chloroform method to extract DNA from peanut leaves. The protocol is as followed:
- Grind peanut leaves in 1% SDS in Tris buffer, incubate at 60 oC for 5 mins. Centrifuge.
- Pour supernatant into a mixture of phenol : chloroform : isoamy alcohol (25:24:1). Gently shake. Centrifuge.
- Carefully move upper layer into isopropanol. Incubate at -20 oC for 30 mins. Centrifuge.
- Wash the pellet with ethanol 70%. Centrifuge.
We failed to remove the pigment from the DNA with this protocol. The resulted DNA pellet was dark green.
We have tried using CTAB, adding activated charcoal, B-Mecaptoethanol, repeat the phenol : chloroform step twice. None worked.
Has anyone had experience with extracting DNA from peanut leaves? Can you give us some suggestion how to deal with the color?
I have never seen a dark green DNA pellet when doing phenol-chloroform extraction for plants, but sometimes I observe a light green color. In this case, you can wash the pellet several times with absolute ethanol or isopropanol until the pigment dissolves and the pellet turns white.
As an alternative, you can soak your leaf samples in absolute ethanol for several days, changing the ethanol daily before doing the DNA extraction. This helps reduce pigment while still preserving the DNA in the sample.
The problem mentioned here indicate that the DNA extraction protocol was not optimized. If there is dark green colour, it is due to the inefficient washing steps. I suggest to optimize the extraction protocol before using actual samples for extraction. Few steps which might improve the process
* Use liquid nitrogen, if possible to save and grind the leaf material
* Use vigorous shaking in the second step of yours
* Optimize centrifuge speed and time
* There are several published protocols available and none mentioned colouration problem which indicate the need of optimization of your protocol
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication