Isolation of cell-free DNA from up to 5 ml of blood plasma
Superior recovery of fragmented cell-free DNA
Parallel purification of 24 samples—special adapter set for NucleoVac 96 Vacuum Manifold
Optimized protocol for Cell-Free DNA BCT (“Streck Tubes”)
NucleoSpin cfDNA Midi is designed for rapid preparation of highly pure cell-free DNA from up to 5 ml of blood plasma using vacuum filtration. The application of vacuum filtration technology eliminates tedious centrifugation steps and manual handling of columns during the process (e.g., disposal of collected column flowthrough). Combining NucleoSpin cfDNA Midi with the NucleoVac 96 Vacuum Manifold and the Starter Set Midi enables processing of up to 24 samples in parallel in ~90 minutes.
The purification protocol is based on the lysis of the plasma samples using Proteinase K incubation in the presence of a chaotropic salt-containing buffer. Following the initial lysis step, a binding buffer is added to the samples and the lysate is loaded onto the binding columns. After loading and vacuum filtration-driven binding of the DNA to the silica membrane, impurities are removed by several washing steps, and cfDNA is eluted after a final drying step (Figure 1).
Figure 1. The NucleoSpin cfDNA Midi procedure.
Application data
Efficient isolation of cfDNA from plasma
The NucleoSpin cfDNA Midi kit was used to isolate cfDNA from 2 ml of human EDTA plasma in comparison with a competitor kit (Figure 2).
Figure 2. Efficient isolation of cfDNA from 2 ml of human EDTA plasma. cfDNA was isolated from individual EDTA plasma samples with the NucleoSpin cfDNA Midi kit and a vacuum-based kit from a competitor (Competitor Q). DNA yields were quantified by qPCR (Quantifiler Human DNA Quantification Kit, Thermo Fisher Scientific on an Applied Biosystems 7500 Real-Time PCR System, Thermo Fisher Scientific). For each sample processed, NucleoSpin cfDNA Midi provided DNA yields that were either greater than or comparable to the yields obtained with the kit from Competitor Q.
Optimized protocol for Cell-Free DNA BCT (“Streck Tubes”)
The NucleoSpin cfDNA Midi kit provided similar yields of cfDNA from blood samples preserved with Cell-Free DNA BCT (“Streck Tubes”) and EDTA tubes, due to optimization of the protocol for the “Streck Tubes” (Figure 3).
Figure 3. Optimized protocol for cfDNA isolation from Cell-Free DNA BCT (“Streck Tubes”). Blood was drawn from five independent test subjects and preserved using both Cell-Free DNA BCT (“Streck Tubes”) and EDTA tubes. cfDNA was isolated from samples preserved in both tube types using the NucleoSpin cfDNA Midi kit. cfDNA yields from EDTA tubes and “Streck Tubes” were similar. DNA yields were quantified by qPCR (Quantifiler Human DNA Quantification Kit, Thermo Fisher Scientific on an Applied Biosystems 7500 Real-Time PCR System, Thermo Fisher Scientific).
Product-at-a-glance
Technology
Silica membrane
Format
Midi spin columns
Starting material
1–5 ml plasma
Blood draw tubes
EDTA, Cell-Free DNA BCT (Streck)
Fragment size
≥50 bp
Typical yield
Varies depending on sample source, storage, and quality
