Samuel J Gurr, PhD

Geoduck DNA RNA extraction

by Samuel J Gurr PhD 8 min read

Geoduck DNA/RNA extractions

Objective(s):

Follow the Zymo Research Quick-DNA/RNA MiniPrep Plus Kit protocol and homogenization steps tested by K. Wong (link below) for geoduck extractions on 20200730

(I) K.Wong’s notebook posts:

(II) Zymo Research Quick-DNA/RNA MiniPrep Plus Kit link here

Samples today:

Geoduck ID # animals Storage Box # Position Experiment day Treatment
219 1 3 A3 4 AHM
234 1 3 E9 4 AHS
282 1 3 D1 4 EHA

Sample tracker of Github repo here link

Geoduck Homogenization

  • Modifications and notes in addition to K.Wong’s tests link here written below in RED
  • I also BOLD text to emphasize transition of samples/reagents to new or different receptacles, this will help me streamline equipment prep in future extractions
  1. Removed samples from -80 °C.
  2. Add 1 mL of RNA/DNA shield
  3. Add 0.25 mL of 0.5mm glass beads to each tube (the same tube from the -80 °C)
  4. Vortex for one minute. Leave the settings on and on max power.
    • Note: must insert the tubes firmly in the vortex or they will fly out. Hover a hand lightly over the samples to prevent this
    • Sample 282 flew out of the vortex and the sample was lost. The remaining processing occuring for samples 219 and 234 only
  5. Remove supernatant from the bead tube and place in a new 1.5 microcentrifuge tube labeled on the side with the extraction sample number and today’s date. Label the cap of the microcentrifuge tube with the sample number. This supernatant will become the sample for “soft homogenization”.
    • 300 μl of supernatant was taken to avoid beads and geoduck shell/tissue
    • Note: attempt to retain more supertatant in future extractions. I vortexed for a shorter period of time here (one minute) to attempt to increase the quality of DNA in downstream analysis
  6. In a new microcentrifuge tube (1.5 ml) add 300 μl of sample, 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to a new 1.5 mL microcentrifuge tube.
    • Important!
    • Proteinase K does not come with the zymo kit find this in the -20°C - summary:
      • 10:1 for Sample:proteinase K digestion buffer
      • 2:1 for proteinase K digestion buffer:Proteinase K </span>
      • the volume(s) here depends on the amount of supernantant taken after soft homogenization
  7. Vortex and spin down all tubes.
  8. Trasfer ### μl of superatant to a new 5 mL tube.
    • Important ‘### μl’ depends on the volume of supernatant taken in step #5
    • today I was able to take 300 μl - this has important infleunce on the volumes used below… </span>

Zymo Research Quick-DNA/RNA MiniPrep Plus Kit

  • Modifications to K.Wong’s tests link here written below in RED
  • I also BOLD text to emphasize transition of samples/reagents to new or different receptacles, this will help me streamline equipment prep in future extractions

DNA Extraction

  1. Set up yellow DNA spin columns and collection tubes, label appropriately
    • n = 1 yellow spin column per sample
  2. Warm elution liquids to 70 °C (10mM Tris HCl pH. 8.0 and RNase free water)
    • Use a Thermomixer to keep the two samples warmed
    • Note: thes solutions are used as the final steps for DNA extraction (Tris) and RNA extraction (RNAse free water)
    • How much 10nM Tris?
      • n * 100 µl Tris == volume needed (review steps # 18 and 21)
      • today… 2 * 100 µl == 200 µl Tris </span>
    • How much RNase free water?
      • n * 100 µl Tris == volume needed (review steps # 18 and 21)
      • today… 2 * 100 µl == 200 µl RNase free water </span>
  3. Add equal volume (to supernatant; ### µl ) DNA/RNA lysis buffer to each sample tube
    • ‘ ### µl’ depends on # 8 in ‘Geoduck Homogenization’
  4. Finger flick to mix tubes
  5. Add half of the total volume of sample gently to the yellow DNA spin column
  6. Centrifuge at 16,000 rcf (g) for 30 seconds
  7. Important if running RNA extraction! Save the flow through from this step: transfer to a new 5 mL tube labeled for RNA
    • Repeat steps 5-7 until all volume is gone. (i.e. x 2)
    • Example: if total volume was 1400 µl, you would run steps 5-7 twice with 700 µl each run
    • Total volume today was 600 µl; this volume will double in RNA extractions below (add equal volume EtOH) today I used 1.5 ml tube here because of the low volume of supernantat retained post-homogenization.
  8. Add 400 µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
  9. Centrifuge at 16,000 rcf (g) for 30 seconds
  10. Discard flow through (Zymo kit waste)
  11. Add 700 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
  12. Centrifuge at 16,000 rcf (g) for 30 seconds
  13. Discard flow through (Zymo kit waste)
  14. Add 400 µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
  15. Centrifuge at 16,000 rcf (g) for 2 minutes
  16. Discard flow through (Zymo kit waste)
  17. Transfer yellow columns to new 1.5 mL microcentrifuge tubes (“E1”)
  18. Add 50 µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter
  19. Incubate at room temperature for 5 minutes. Centrifuge at 16,000 rcf (g) for 30 seconds keep the flow through
  20. Add another 50 µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter
  21. Incubate at room temperature for 15 minutes.
  22. Centrifuge at 16,000 rcf (g) for 30 seconds
  23. Throw away filter and keep flow through.
  24. Aliquot 10 µl of the elution to 0.5 mL PCR tubes for Qubit and Gel Electrophoresis analysis.
    • n = 1 tube per sample
  25. Store at 4 °C if quantifying the same day or the next, if waiting longer store in -20 °C freezer.

RNA Extraction

Can do concurrently with DNA Extraction after DNA Extraction Step 7

  1. Add equal volume ( Total volume today was 600 µl - review step # 7 ) 100% EtOH to the tubes labeled for RNA containing the original yellow column flow through

Note: Tube size used today was 1.5 ml to hold the 1200 µl total volume after this step. If/when more supernatant is taken (# 5 in ‘Geoduck Homogenization’), you will require a 5ml tube in step # 7 of ‘DNA extraction’!

  1. Vortex and spin down to mix
  2. Add 700 µl of that liquid to the green RNA spin columns
  3. Centrifuge at 16,000 rcf (g) for 30 seconds
  4. Discard flow through (Zymo kit waste)
  5. Add 700 µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
  6. Centrifuge at 16,000 rcf (g) for 30 seconds
    • Repeat until all volume is gone (i.e. x 4)
    • Get DNase I from freezer

Note: steps # 3- 7 were written assuming 2800 µl total volume after step 1. Today I had 1200 µl total volume after step 1 so I ran this X 2 for 600 µl each run.

  1. Discard flow through (Zymo kit waste)
  2. Add 400 µl DNA/RNA Wash Buffer gently to each green RNA column
  3. Centrifuge at 16,000 rcf (g) for 30 seconds
  4. Discard flow through (Zymo kit waste)
  5. Make DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples

Today I had 2 samples, therefore: 150 µl of DNA Digestion buffer and 10 µl of DNase I

  1. Add 80 µl DNase I treatment master mix directly to the filter of the green RNA columns
  2. Incubate at room temp for 15 minutes
  3. Add 400 µl DNA/RNA Prep Buffer gently to each column
  4. Centrifuge at 16,000 rcf (g) for 30 seconds
  5. Discard flow through (Zymo kit waste)
  6. Add 700 µl DNA/RNA Wash Buffer gently to the green RNA spin columns
  7. Centrifuge at 16,000 rcf (g) for 30 seconds
  8. Discard flow through (Zymo kit waste)
  9. Add 400 µl DNA/RNA Wash Buffer genetly to the green RNA spin columns
  10. Centrifuge at 16,000 rcf (g) for 2 minutes
  11. Discard flow through (Zymo kit waste)
  12. Transfer green columns to new 1.5 mL microcentrifuge tubes
  13. Add 50 µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filter
  14. Incubate at room temp for 5 minutes
  15. Centrifuge at 16,000 rcf (g) for 30 seconds
  16. Repeat steps 25-27 for a final elution volume of 100 µl
  17. Aliquot 5 µl of the final elution to 0.5 mL PCR tubes for Qubit and TapeStation analysis.
    • n = 1 tube per sample
  18. Store all tubes in the -80 °C freezer.

Clean-up

  1. Place tissue and liquid in the waste container labeled Zymo extraction waste.
  2. Wipe down RNA free area with RNase away and kimwipes.
  3. Throw away all tips and restock tip boxes if necessary.

Testing Quantity and Quality

Test type DNA and/or RNA Instrument Completed today (Y/N)
Quantity RNA + DNA Qubit; click here for link protocol Y
Quality RNA only TapeStation; click here for link protocol N
Quality DNA only Gel Electrophoresis; click here for link protocol N

Note: The application of these instruments for DNA and/or RNA is in reference to the materials we currently have available in the lab - i.e. there are alternative materials/reagents to run gel electrophoresis for RNA

Quantify Results (only Qubit ran today)

Qubit

2 samples + 2 standards + 1 error = 4

DNA Broad Range

199 µl Buffer x 4 = 796 µl Buffer 1 µl Reagent x 4 = 4 µl Reagent

RNA Broad Range

199 µl Buffer x 4 = 796 µl Buffer 1 µl Reagent x 4 = 4 µl Reagent

Sample DNA (ng/uL)   RNA (ng/uL)  
  RUN 1 RUN 2 RUN 1 RUN 2
Standard 1 192.16 194.61 388.6 385.81
Standard 2 21864.16 21060.07 9533.43 9484.34
219 27.2 29.4 31.8 31.8
234 12.3 12.0 75.2 73.2

For Further Analsis…

0.5 mL PCR tubes aliquots for Tapestation and gel electrophoresis located in -80C fridge, geoduck storage box # 3