Zymo Quick-DNA Mini Prep Kit Troubleshooting on Adult P. astreoides

Troubleshooting Zymo Quick-DNA Mini Prep kit on adult P. astreoides homogenates.

Experimental design

Table 1: BIOS 2017 adult homogenate vials used for this troubleshooting.

Sample # Sample Type Coral ID/Vial# Volume Prepared by
7 Homogenate 7 500 mL Kevin
16 Homogenate 16 500 mL Erin
25 Homogenate 25 500 mL Erin
34 Homogenate 34 500 mL Kevin

Protocol

  • Using slightly modified (Below are summary steps)

Equipment

  • Incubator that can be set to 55°C
  • Tabletop centrifuges for 1.5mL tubes capable of 12,000 x g

Enzyme preparation

Protenase K enzymes in the kit come in dry/powdered form, therefore it must be re-hydrated with the Protenase K storage buffer

5 mg of Protenase K solution + 265 uL of Protentase K storage buffer

Sample Preparation

  • ** Warm the DNA Elution Buffer to 55°C before starting**

  • Following the Biological fluids and cell protocol
  • Let vials thaw
  • Spin down homogenate on small table top centrifuge for 2 mins
  • Remove 200 uL of supernatant and transfer into new 1.5 mL tubes

DNA Extraction

  1. Add 200 uL Biofluid Cell Buffer (red) and 20uL Protenase K to each 1.5 mL tube
    • Mix with vortex and quick spin on small table top centrifuge
  2. Incubate in Thermomixer at 55°C speed 600 for 10 minutes
  3. Add equal volume (420 uL) of Genomic Binding Buffer to each 1.5 ml tube
    • Mix with vortex and quick spin on small table top centrifuge
  4. Add the total volume (840 uL) of the 1.5 mL tube to the spin column
    • Centrifuge at 12,000 g (rcf) for 1 minute
  5. Transfer spin column to a new column (discard previous column)
  6. Add 400 uL pre-wash buffer to each spin column
    • Centrifuge at 12,000 g (rcf) for 1 minute
  7. Remove and discard flow through
  8. Add 700 uL pre-wash buffer to each spin column
    • Centrifuge at 12,000 g (rcf) for 1 minute
  9. Remove and discard flow through
  10. Add 200 uL wash buffer to each spin column
    • Centrifuge at 12,000 g (rcf) for 2 minute
  11. Transfer spin columns to new 1.5 mL centrifuge tubes
  12. Vortex and spin DNA Elution Buffer
  13. Add 50 uL of DNA Elution Buffer directly to the filter in the spin column
    • Incubate at room temperature for 5 minutes
  14. Centrifuge at maximum speed (21130 rcf) for 1 minute
  15. Add another 50 uL of DNA Elution Buffer directly to the filter in the spin column
    • Incubate at room temperature for 3 minutes
  16. Centrifuge at maximum speed (21130 rcf) for 1 minute
  17. Label final tubes
  18. If quantifying immediately, place in 4deg;C fridge
  19. If quantifying later, aliquot appropriately and store in -80deg;C

Quantification

Quibit

| | Standard 1 | Standard 2 | 7 (ng/uL) | 16 (ng/uL) | 25 (ng/uL) | 34 (ng/uL) | |:—:|:———-:|:———–:|:———:|:———-:|:———-:|:———-:| | DNA | 168.52 | 18228.46 | Too Low | 3.68 | 4.60 | Too Low | | DNA | NA | NA | Too Low | 3.74 | 4.48 | Too Low |

Gel

| | Well 1 | Well 2 | Well 3 | Well 4 | Well 5 | Well 6 | Well 7 | Well 8 | Well 9 | |:——:|:——:|:——:|:——:|:——:|:——:|——–|:——:|——–|———| | Sample | Ladder | 1 | 2 | 3 | 4 | 7 | 16 | 25 | 34 |

Results

  • DNA did not show up well in gel, possibly because of too low of concentrations
  • The gel was a bit thicker than usual (>100mL), therefore could have contributed to lighter bands

Troubleshoot

  • Need to concentrate samples
    • Possibly vacufuge sample to concentrate it to 200uL
Written on March 3, 2019