38 Preparation
Review the figures below to learn about how the New England BioLabs Monarch High Molecular Weight (HMW) DNA Extraction kit works.
Procedure
Bacterial Lysis and Genomic DNA Isolation
Each group will need the items listed below (source: New England BioLabs Monarch High Molecular Weight DNA Extraction Kit Protocol).
Bacterial Lysis:
- RNase A, 10 μl per sample (provided in kit), and Proteinase K (50 mg/ml), 20 μl per sample
- Stored on ice for the duration of the procedure.
- When in use, keep the vials in or near the surface of the ice as much as possible. Both substances are very temperature sensitive, and warming them will negatively affect your results.
- Cold PBS or 10 mM Tris buffer, 300 μl per sample
- HMW gDNA Tissue Lysis Buffer, 300 μl per sample
- Lysozyme solution, 10 μl per sample
- Protein Separation Solution, 300 μl per sample
HMW gDNA Binding and Elution:
- Isopropanol, 550 µl per sample
- gDNA Wash Buffer, 1000 μl per sample
- Add ethanol (≥ 95%) as indicated on the bottle label. (This has most likely already been done for you. Ask your lab instructor if you are unsure.)
- gDNA Elution Buffer II, 100 μl per sample
- DNA Capture Beads
- Bead Retainer, 1 per sample
- Collection Tube II, 1 per sample
- 1.5 ml DNase-free, low DNA binding microfuge tubes, 1 per sample
- 2 ml centrifuge tubes (2 per sample)
- Clean forceps (DNA Away and ethanol-treated
