The Monarch Genomic DNA Purification Kit is a comprehensive solution for cell lysis, RNA removal, and purification of intact genomic
DNA (gDNA) from a wide variety of biological samples, including cultured cells, blood, and mammalian tissues. Additionally, bacteria
and yeast can be processed with extra steps to enhance lysis in these tough-to-lyse samples. Protocols are also included to enable
purification from clinically-relevant samples such as saliva and cheek swabs as well as rapid cleanup of previously extracted gDNA.
Purified gDNA has high quality metrics, including A260/A280 > 1.8 and A260/A230 > 2.0, high DIN scores and minimal residual RNA. The
purified gDNA is suitable for downstream applications such as end-point PCR, qPCR and library prep for NGS. It typically has
a peak size of > 50kb, making this kit an excellent choice upstream of long-read sequencing platforms.
Workflow for the Monarch Genomic DNA Purification Kit
Specifications:
Input:
Cultured mammalian cells: up to 5 x 106 cells
Mammalian whole blood: 100 µl
Tissue: up to 25 mg, depending on tissue type
Bacteria: up to 2 x 109
Yeast: up to 5 x 107
Saliva: up to 500 µl
Buccal swabs
Genomic DNA requiring cleanup
Binding Capacity:
30 µg genomic DNA
Yield:
Varies depending on sample type
Genomic DNA Size:
Peak size > 50 kb for most sample types; may be lower for saliva and buccal swabs
RNA Content
< 1% (with included RNase A treatment)
Elution Volume
≥35 µl, but 100 µl is recommended
Purity:
A260/280 ≥ 1.8
A260/230 ≥ 2.0
Validated Sample Types:
Mouse Tail
Mouse Ear
Mouse Liver
Rat Liver
Mouse Kidney
Mouse Spleen
Mouse Heart
Mouse Lung
Mouse Brain
Rat Brain
Mouse Muscle
Rat Muscle
Deer Muscle
Human Blood
Mouse Blood
Rabbit Blood
Pig Blood
Guinea Pig Blood
Cow Blood
Horse Blood
Dog Blood
Chicken Blood
HeLa Cells
HEK293 Cells
NIH3T3 Cells
E. coli
Rhodobacter sp.
B. cereus
T. kodakarensis
S. cerevisiae
Saliva
Buccal swab
Figure 1: The Monarch Genomic DNA Purification Kit efficiently purifies high-quality, high molecular weight gDNA from a
variety of sample types.
100 ng of genomic DNA from each sample was loaded on a 0.75% agarose gel. gDNA was isolated following the standard protocols for
blood, cultured cells and tissue, and the supplemental protocols for buccal swabs, saliva, Gram– and Gram+ bacteria. Starting material
used: 1 x 106 HeLa cells, 100 μl human blood, 10 μl bird blood, 10 mg frozen tissue powder, 1 buccal swab, 500 μl saliva and ~1 x 109 bacterial cells. Lambda DNA-Hind III digest (NEB #N3012) was used as a marker in the last lane (M). Purified gDNA samples were
analyzed using a Genomic DNA ScreenTape® on an Agilent Technologies® 4200 TapeStation®. Samples typically yield peak sizes 50–70 kb
and DINs of ~9. The cell fractions processed in the buccal swab and saliva preps contain dead cells, as expected, causing a smear like
pattern with typical low molecular weight apoptotic bands.
Figure 2: The Monarch Genomic DNA Purification Kit provides excellent yields for tissues that are problematic for other commercial kits.
Duplicate 10 mg samples of RNAlater®-stabilized rat tissue were cut to small pieces and subsequently lysed and purified according to the protocols provided for each kit. Optional RNase A steps were included. Elution was carried out with 100 µl elution buffer provided in the respective kits. Yields displayed are averages of the duplicate samples, and represent the genomic DNA yield after correcting for the RNA content as determined by LC-MS. Results indicate that the Monarch Genomic DNA Purification Kit provides excellent yields for a wide range of tissues.
Figure 3: Genomic DNA purified with the Monarch Genomic DNA Purification Kit is high quality and suitable for sensitive applications like long range PCR and qPCR.
A. Amplification reactions were set up with primer pairs specific for 6, 8, 10, 12, 16, 20 kb amplicons from human DNA. LongAmp® Hot Start Taq 2X Master Mix (NEB #M0533) was used and 25 ng template DNA was added to each sample. PCR reactions were carried out on an Applied Biosystems 2720 Thermal Cycler. Monarch-purified genomic DNA isolated from HeLa cells and human blood were compared to commercially available reference DNA from the human cell line NA19240 F11. 10 of 20 µl was loaded on a 1.5% agarose gel, using the 1 kb DNA Ladder (NEB #N3232) as a marker. Results indicated DNA was of high-integrity and suitable for long range PCR.
B. Monarch-purified genomic DNA from human whole blood, HeLa cells and mouse tail was diluted to produce a five log range of input template concentrations. The results were generated using primers targeting gHEME (human whole blood) and gREL (HeLa, mouse tail) for qPCR assays with the Luna Universal qPCR Master Mix (NEB #M3003) and cycled on a Bio-Rad® CFX Touch qPCR thermal cycler. Results indicated that DNA is highly pure and free from inhibitors, optimal for qPCR.
Figure 4: The Monarch Genomic DNA Purification Kit produces excellent input material for NGS library preparation with NEBNext® kits for Illumina®.
A. Duplicate libraries were made from 100 ng HeLa cell gDNA purified with Monarch (orange) or Qiagen DNeasy Mini Kit (blue) using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (NEB #E7805). Libraries were sequenced on an Illumina MiSeq. Reads were mapped using Bowtie 2.2.4 and GC coverage was calculated using Picard’s CollectGCBiasMetrics (v1.117). Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each %GC is indicated by the vertical grey bars, and the colored lines represent the normalized coverage for each library. Monarch GC coverage matched Qiagen DNeasy results.
B. Yields of libraries produced by enzymatic shearing are equivalent when using Monarch-purified gDNA. Library yields of the samples described above were assessed on an Agilent Technologies® 2100 BioAnalyzer using a High Sensitivity DNA Kit.
Figure 5: DNA isolation from higher volume and amounts of blood using RBC Lysis based protocol for Monarch gDNA Purification Kit
DNA extracted using RBC lysis method achieves similar high yields and concentrations as direct methods, while allowing for extraction from higher volumes and amounts of blood. Genomic DNA samples were purified in triplicate from 3 days old fresh pig blood. For the Direct Lysis samples, the DNA from 100 µl pig blood was purified using the standard protocol for fresh mammalian blood from the NEB #T3010 kit. For the RBC Lysis samples, the indicated amount of pig blood was used in the protocol for up to 500 µl blood using the NEB #T3010 kit and RBC Lysis Buffer (NEB #T3051).
Figure 6: The Monarch Genomic DNA Purification Kit generates high quality DNA for nanopore sequencing.
HeLa cell genomic DNA was extracted using either the Monarch Genomic DNA Purification Kit or the Qiagen DNeasy Blood & Tissue Kit. One microgram of purified DNA was used to prepare Oxford Nanopore Technology (ONT) sequencing libraries following the ONT 1D Ligation Sequencing Kit (SQK-LSK109) protocol without DNA fragmentation. Libraries were loaded on a GridION (Flow cell R9.4.1) and the data was collected for 48 hrs. Libraries produced using the Monarch gDNA exceeded the Qiagen libraries on common sequencing metrics including: A. total sequencing data collected, B. read quality, and C. read length. Data was generated using NanoComp (Bioinformatics, Volume 34, Issue 15, 1 August 2018, Pages 2666–2669).
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