Hello,
I have a problem getting clean DNA from my DNA extractions, I have tried multiple times and consulted the extraction kit manufacturer without any particular luck. According to the Nanodrop spectrophotometer, the yields and absorption values are as follows:
Sample_ID; Nucleic_Acid_Conc; A260; A280; 260/280; 260/230
1; 7.6 ng/µl; 0.152; 0.086; 1.76; 0.71
2; 24.0 ng/µl; 0.479; 0.249; 1.93; 1.16
3; 21.9 ng/µl; 0.439; 0.238; 1.84; 1.15
4; 18.6 ng/µl; 0.371; 0.190; 1.95; 1.29
5; 18.5 ng/µl; 0.371; 0.200; 1.85; 1.44
6; 6.9 ng/µl; 0.138; 0.069; 2.00; 0.70
7; 7.9 ng/µl; 0.157; 0.092; 1.72; 0.78
8; 35.2 ng/µl; 0.703; 0.363; 1.94; 1.67
9; 9.6 ng/µl; 0.192; 0.099; 1.94; 0.85
extraction_blank; 1.6 ng/µl; 0.031; 0.006; 4.84; 0.19
collection_blank; 3.9 ng/µl; 0.078; 0.036; 2.19; 0.42
Settings at "DNA", factor 50. Total extraction volume 100 µl, so total DNA yields are the concentrations multiplied by 100.
The question now is whether this is safe to proceed with. According to ThermoFisher "good" values are between 1.8-2.0 for the 260/280 ratio, and 2.0-2.2 for the 260/230 ratio. My samples seem generally ok for the 260/280 ratios (exluding the blanks), but the 260/230 ratios are consistently lower by quite a bit. Is this a problem? I want to do DNA metabarcoding through Illumina MiSeq sequencing of a couple of marker genes. This means I will do PCR following the DNA extraction, so on the one hand I won't need so much DNA extract. On the other hand, the PCR may be the critical step, where contaminants can interfere. Maybe it even risks spoiling the sequencing run? What are your opinions? How do you treat your own samples based on spectrophotometer values?
I have a problem getting clean DNA from my DNA extractions, I have tried multiple times and consulted the extraction kit manufacturer without any particular luck. According to the Nanodrop spectrophotometer, the yields and absorption values are as follows:
Sample_ID; Nucleic_Acid_Conc; A260; A280; 260/280; 260/230
1; 7.6 ng/µl; 0.152; 0.086; 1.76; 0.71
2; 24.0 ng/µl; 0.479; 0.249; 1.93; 1.16
3; 21.9 ng/µl; 0.439; 0.238; 1.84; 1.15
4; 18.6 ng/µl; 0.371; 0.190; 1.95; 1.29
5; 18.5 ng/µl; 0.371; 0.200; 1.85; 1.44
6; 6.9 ng/µl; 0.138; 0.069; 2.00; 0.70
7; 7.9 ng/µl; 0.157; 0.092; 1.72; 0.78
8; 35.2 ng/µl; 0.703; 0.363; 1.94; 1.67
9; 9.6 ng/µl; 0.192; 0.099; 1.94; 0.85
extraction_blank; 1.6 ng/µl; 0.031; 0.006; 4.84; 0.19
collection_blank; 3.9 ng/µl; 0.078; 0.036; 2.19; 0.42
Settings at "DNA", factor 50. Total extraction volume 100 µl, so total DNA yields are the concentrations multiplied by 100.
The question now is whether this is safe to proceed with. According to ThermoFisher "good" values are between 1.8-2.0 for the 260/280 ratio, and 2.0-2.2 for the 260/230 ratio. My samples seem generally ok for the 260/280 ratios (exluding the blanks), but the 260/230 ratios are consistently lower by quite a bit. Is this a problem? I want to do DNA metabarcoding through Illumina MiSeq sequencing of a couple of marker genes. This means I will do PCR following the DNA extraction, so on the one hand I won't need so much DNA extract. On the other hand, the PCR may be the critical step, where contaminants can interfere. Maybe it even risks spoiling the sequencing run? What are your opinions? How do you treat your own samples based on spectrophotometer values?