I have extracted DNA from cacao leaf tissue (freeze dried).
I used CTAB buffer (5%) along with 1% B-mercaptoethanol and 3% PVP. I did the DNA extraction overseas and transported it as a pellet to Australia. When they arrived in Australia I stored them at 4 C after resuspending them in TE buffer. However, when I checked the quality on agarose gel surprisingly all of my samples have degraded DNA.
Could anyone pls advice me what can I do to prevent the degradation of the DNA? The downstream application of the DNA will be DarT analysis.
I have attached the gel picture as well.
Regards
Gurpreet
Dear Gurpreet,
Are you using DNase free tubes and are you sure your buffers are clean?
Do I see it correctly that the first sample is still okay?
I would recommend not sending your DNA as a pellet but as freeze dried sample or as the isolated DNA in buffer.
Hi Silvia, I am not sure if they were DNAse free. Do I need sterilise the buffer and tubes prior to extraction?
Hi Gurpreet,
You can autoclave your eppendorf tubes before use. Do not autoclave buffers containing SDS or B-mercaptoethanol ;-).
The key steps to prevent DNA degradation are:
Hi Gurpreet,
Perhaps the best way is to re-extract the DNA again from the fresh and young cacao leaves. If you were to repeat the DNA extraction, you may consider to grind in less leaves sample (maybe 20 mg will do). However, if you cannot repeat the DNA extraction, you can perhaps to run a general PCR using chloroplast primers to confirm the presence of your targeted DNA before you proceed with downstream analysis.
I hope it helps! All the best!
Hi Kok Sim,
I cannot extract from young and fresh leaves as the leaves were collected overseas. I have the tissue preserved in 96% ethanol. I initially used 120 mg of the leaf tissue. Do you think this is degraded DNA or the smears are just from the impurities?
Hi Gurpreet,
I think the smearing is due to RNA contamination. The reason genomic DNA is not observed on top could likely due to low concentration of DNA extracted that is extracted from the leaves.
Hi Gurpreet,
I suggest u to re-precipitate the sample in isopropanol or absolute ethanol with full tube and then pellet down the DNA. I am sure, u will get the DNA
All the Best...
I think you DNA degraded/shearing during transportation via high temperature and mechanical stress. A better way is try using DMSO on your DNA to see if it will work. To prevent is to do your extraction and analysis in same place or maintain -4 degrees tempt during transportation.
Using proteinase is a very crucial step in DNA isolation since it will purify your extract from the nuclease proteins. Moreover perform your isolation in low temperature since it will inhibit remained nuclease activity. So if you have done all steps correctly (handling & storage of starting material) please be sure about your proteinase functionality.
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