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Before you begin the protocol, please make sure you are equipped with the appropriate lab equipment and reagents listed below. 

Additionally, please consult the provided sample input tables, as well as the protocol variation options to make sure no additional reagents are needed before beginning the prep. Do not perform any additional mechanical lysis other than what is described in the protocol using a bioruptor or similar instrument. This will very likely result in a failed library prep. If you have any further questions, please consult our FAQ or reach out to support@phasegenomics.com

 

Needed Equipment and Reagents:

Reagents

The following molecular-biology grade reagents are required to complete this protocol. Ensure that reagents are free of DNA, RNA and nucleases.

  • 80% Ethanol
  • Deionized or nanopure water

Equipment and Consumables

No specialist equipment is required for this protocol. The following general laboratory equipment and consumables are needed: 

  • Calibrated 2 – 10 μL pipette and filtered tips
  • Calibrated 10 – 100 μL pipette and filtered tips
  • Calibrated 200 – 1000 μL pipette and filtered tips
  • 1.5 or 2 mL microcentrifuge tubes
  • 0.2 mL PCR tubes
  • Magnetic tube rack/magnet for 2 mL microcentrifuge tubes or 0.2 mL PCR tubes (depending on tube type used in step 2.8).
  • Microcentrifuge capable of ≥17,000 x g
  • Thermocycler
  • Heating block or water bath that can maintain a temperature of 65˚C (alternatively use a thermocycler)
  • Vortexer
  • Qubit Fluorometer and Qubit dsDNA DNA HS Assay Kit (Thermo Fisher Scientific), or similar fluorometric assay for the quantification of double-stranded DNA

Sample Input Requirements:

In general, fresh, frozen, -80°, and liquid nitrogen are acceptable for sample storage. RNAlater™, ZymoShield™, or ethanol are also acceptable for sample storage (room temp storage OK). 

 

Sample
Standard Project Inputs

Animal

Tissue (200-300 mg)

Blood 200-300 µL)

Insects (200-300 mg)

Cells (1-5 M)

Plant

Leaf (200-300 mg)

Seedling tissue (> 250 mg)

Flower tissue (>250 mg)

High complexity community

>200 species/strains expected (e.g. soil)

>20g raw sample

 

Medium complexity community

20-200 species/strains (e.g. fecal)

50-200 µL raw sample

 

Low complexity community

<20 species/strains expected (e.g. culture)

50-200 µL raw sample

 

Cells

<20 species/strains expected (e.g. culture)

>1 million cells

 

 

Preservation Method
Standard Preservation Conditions

Fresh

Still living within 48 hours of library preparation

Frozen

Flash frozen

Stored at -80˚C for < 1 year

Sample freeze-thawed < 2x

Preserved

<1 month at room temperature

Stored in RNAlater, Ethanol, or Zymo-Shield

Fixed tissue

<2% formaldehyde for 10-20 minutes, then quenched

 

Sample Type
Suggested Protocol Variation

Animal Soft Tissue

Instead of liquid nitrogen grinding, use dounce homogenizer in 700 µL Animal Lysis Buffer 1

Cells or Whole Blood

No mechanical lysis needed; proceed directly to step 2.2

Soil samples

See Proximo (microbe) appendices A1-2

 

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