Pour une purification rapide et simple de l'ADN bactérien et de l'ADN de l'hôte à partir d'échantillons de selles.
Détails
Supporting Data
Figure 1. Higher Yields of DNA than Competitor Q.
Stool DNA was isolated from 200 mg of fresh or preserved stool samples using Norgen’s Stool DNA Isolation Kit and Competitor Q's Kit. For evaluation, 10 µL of DNA from the elution was run on 1X TAE 1.2% agarose gel. Norgen's kit isolated much higher yields of DNA. *Stool was collected using Norgen’s Stool Nucleic Acid Collection and Preservation tubes (Cat. 45660). Marker = Norgen’s HighRanger DNA Ladder (Cat. 11900).
Figure 2. Stool DNA quality and concentration measured by NanoDrop.
High DNA concentration and quality was obtained using Norgen’s Stool DNA Isolation Kit from fresh or preserved stool samples. Figure 2A: Fresh stool samples, Figure 2B: Preserved stool using Norgen’s Stool Nucleic Acid Collection and Preservation Tubes (Cat. 45660).
Figure 3. Detection of 16S rRNA from stool DNA isolated from 200 mg of fresh stool samples using Norgen's Stool DNA Isolation Kit and Competitor Q’s kit.
DNA quality was confirmed by Real-time PCR using 2 µL of stool DNA (total PCR reaction volume was 20 µL) to detect 16S rRNA from different microorganisms in the stool samples. The earlier Ct value with Norgen's DNA samples (blue lines) compared to Competitor Q’s samples (red lines) indicated a higher quality of stool DNA for downstream applications. Figure 3A: Fresh stool sample, Figure 3B: Preserved stool using Norgen’s Stool Nucleic Acid Collection and Preservation Tubes (Cat. 45660).
Figure 4. Detection of 5S rRNA from the Stool DNA isolated from 200 mg of fresh stool samples using Norgen's Stool DNA Isolation Kit and Competitor Q’s Kit.
DNA quality was confirmed by Real-time PCR using 2 µL of stool DNA (total PCR reaction volume was 20 µL) to detect 5S rRNA from eukaryotic DNA in the stool samples. The earlier Ct value with Norgen's DNA samples (blue lines) compared to Competitor Q’s samples (red lines) indicated a higher quality of stool DNA for downstream applications. Figure A: Fresh stool sample, Figure B: Preserved stool using Norgen’s Stool Nucleic Acid Collection and Preservation Tubes (Cat. 45660).
Figure 5. Better 16S rRNA detection from DNA isolated using Norgen’s Stool DNA Preservative and Stool DNA Isolation Kit.
Two microlitres of stool DNA was used in a 20 µL PCR reaction volume. Stool was preserved using Norgen’s Stool DNA Preservative and isolated using Norgen’s Stool DNA Isolation Kit showed better 16S rRNA gene detection as compared to fresh stool isolated with Competitor Q.
Figure 6. Distribution of 10 Different Fecal Microbiomes.
A) Principal Coordinate Analysis of 10 fecal microbiomes showing differences in the distribution of taxonomic classifications between samples up to class level. B) Hierarchical clustering of 10 fecal microbiomes based on genus-level classifications, including a bar chart showing the relative abundance of genus-level classifications for each sample in the dendrogram.
Figure 7. High Quality DNA Isolated from Preserved Stool Samples.
DNA was isolated from 200 µL preserved stool samples using Norgen’s Stool DNA Isolation Kit (Magnetic Bead System).For evaluation, 10 µL from 75 µL of elution were run on 1X TAE 1.2% agarose gel. Stool samples were preserved in Norgen’s Stool Nucleic Acid Collection and Transport Tubes (Cat. 45630, 45660). M = Norgen’s HighRanger DNA Ladder (Cat. 11900).
Figure 8. Comparison of A260/280 and A260/230 ratios.
DNA was isolated from 200 mg stool samples using Norgen's Stool DNA Isolation Kit (Magnetic Bead System) and Norgen's Stool DNA Isolation Kit (column format). The purified DNA was then compared for A260/280 and A260/230 ratios. DNA isolated using Norgen’s Stool DNA Isolation Kit (Magnetic Bead System) (A, B and C) showed a comparable DNA quality to the DNA isolated using Norgen’s Stool DNA Isolation (Column method), indicating the consistence and highest DNA quality were generated by the Stool DNA Isolation Kit (Magnetic Bead System).
Figure 9. High Quality DNA confirmed by Real-time PCR.
DNA was isolated from 200 mg stool using Norgen's Stool DNA Isolation Kit (Magnetic Bead System) and Norgen's Stool DNA Isolation Kit (column format). The quality of the purified DNA was evaluated by using 8 μL of stool DNA (total PCR reaction volume was 20 µL) to detect 16S rRNA from the different stool samples. No PCR inhibition was observed from DNA isolated using Norgen's Stool DNA Isolation Kit (Magnetic Bead System) (blue circles), similar to the DNA isolated using Norgen’s Stool DNA Isolation Kit (Column method) (red circles), indicating the excellent isolation consistency and the quality of the stool DNA for downstream applications.
Figure 10. 16S Metagenomics data generated by Illumina MiSeq.
Stool DNA was isolated using Norgen’s Stool DNA Isolation Kit (Magnetic Bead System) from 200 mg of stool from healthy donors, and the stool microbiomes were successfully sequenced by Illumina MiSeq.
Figure 11. High Quality DNA Isolated from Preserved Stool Samples.
DNA was isolated from 200 µL preserved stool samples using Norgen’s Stool DNA Isolation 96-Well Kit (Magnetic Bead System). For evaluation, 10 µL from 75 µL of elution were run on 1X TAE 1.2% agarose gel. M = Norgen’s HighRanger DNA Ladder (Cat. 11900).
Figure 12. High Quality DNA Confirmed by Real-Time PCR.
DNA was isolated from 200 mg stool using Norgen's Stool DNA Isolation 96-Well Kit (Magnetic Bead System). The quality of the purified DNA was evaluated by using 2 µL of stool DNA (total PCR reaction volume was 20 µL) to detect 16S rRNA from the stool sample. No PCR inhibition was observed from DNA isolated using Norgen's Stool DNA Isolation 96-Well Kit (Magnetic Bead System) indicating the excellent isolation consistency and the high quality of the stool DNA for downstream applications.
Figure 13. Comparison of DNA yield and integrity using two automated platforms with the Stool DNA Isolation 96-Well Kit (High Throughput Magnetic Bead System, Cat. 63100).
DNA was extracted from 200 µL preserved stool samples using the same extraction kit on two automated platforms: Isopure 96 and KingFisher. Total DNA yield was assessed by 16S rRNA gene qPCR (left panel), showing comparable performance across platforms and replicates. Error bars represent standard deviations. High-molecular-weight DNA was consistently recovered from both platforms, demonstrating effective and reproducible DNA isolation regardless of automation system used.
Données justificatives
|
Spécifications du kit
|
|
|
Apport maximal de selles
|
200 mg (selles fraîches/congelées) ou 400 μL (selles conservées)
|
|
Type de selles traitées
|
Selles congelées, fraîches ou conservées
|
| Format | Colonne de Spin |
|
Capacité de liaison maximale de la colonne
|
50 μg
|
| Volume maximal de chargement de la colonne |
650 μL
|
| Volume d'élution/td> | 50 μL |
| Temps nécessaire pour effectuer 10 purifications |
30 minutes
|
| Applications |
PCR, qPCR, analyse de transfert de Southern, séquence, analyse de microréseaux. |
Conditions de stockage et stabilité du produit
Toutes les solutions doivent être maintenues hermétiquement fermées et conservées à température ambiante. Ce kit est stable pendant 2 ans après la date d'expédition.