Hello everyone,
I have extracted microbiome DNA using the Qiamp Powersoil pro kit. After extracting DNA I used nanodrop to check the quality. I used the same last solution present in the DNA to make the blank.
Result:
1. DNA concentrations are around 5 ng/ul
2. 260/280 ratio is 1.27
3. 260/230 ratio is 0.27
Questions:
1. Is the DNA concentration acceptable or too low for microbiome sequencing or qPCR analysis?
2. What could be the reason for the poor 260/280 and 260/230 ratio
3. In the nanodrop graph, you can see no peak but almost a flat curve. What can be the reason for this? (I have attached the graph here)
Please let me know your suggestion regarding this issue. Thanks a lot for your help.
1. Why can't you try with the QIAamp Fast DNA Stool Mini Kit from Qiagen.
2. 5 ng/ul is not good enough but it can be worked so first try with cPCR with gene specific primer rather than adapter ligated primers.
3. amples might be contaminated with chemical components from the kit , poor washing step leads to ethanol residues etc.
4. Poor ratio some tims happens while using kits also low yield of nucleic acid etc.
If you are able to amplify the 16S rRNA then don't worry about yield and purity unless you have a plan to outsource library preparation and sequencing.
Dear Prabhugouda Siriyappagouder ,
Thanks a lot for your answer.
- I tried 16S rRNA-specific primer. It can work. Sharp single band and ct is around 22.
- "unless you have a plan to outsource library preparation and sequencing."
So can I use this sample for 16s rRNA amplicon sequencing?
- My other question is, what concentration do you usually expect after DNA extraction from fish faeces?
Thank you very much.
Regards.
Monzur
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