Zymo-DNA-RNA-Extract-P.astreoides-Genome

RNA/DNA extractions for P. astreoides genome samples

Kit: ZYMO Quick-DNA/RNA Miniprep Plus Kit

Goal

  • To extract DNA and RNA for an Illumina RNAseq library prep workshop

Experimental design

Table 1: Sample descriptions for this extraction

Sample # Sample Type Coral ID Size of Sample Treatment
1 Fragment PG #1 ~10mm diameter 23°C + Air
2 Fragment PG #2 ~10mm diameter -22°C + Dark
  • I sampled duplicates of each sample, preserved them in 500μL DNA/RNA shield in ZR BBashing lysis tubes with 0.1mm and 0.5mm beads and stored at -80°C

Overall workflow

  1. Prepare samples
  2. Extract and prep DNA with column (elute RNA and Protein)
  3. Extract and prep RNA with column (elute Protein)
  4. Quantify DNA, RNA, and protein extracts

Protocol preparation

Reagents and supplies

  • RNase-free Water
  • 100% ethanol, ACS grade or better
  • 10mM Tris HCl pH 8.0 made with RNase-free water
  • ZR BBashing lysis tubes with 0.1mm and 0.5mm beads

Equipment

  • Rocking oven that can be set to 70°C
  • RNase away and a designated RNase free space
  • Tabletop and larger centrifuges for 1.5mL and 50mL tubes capable of 12,000 x g

Clipper Sterilization

  • Rub down clippers with:
    1. 10% Bleach solution
    2. DI water
    3. 90% ethanol
    4. RNAse free water

Notes before starting

  • Wipe down benchtop with RNase away and have the spray bottle and kimwipes on-hand to use frequently

Sample preparation

Fragment preparation

  • Sterilize clippers (as outlined above)
  • Clip off ~10mm in diameter of tissue and skeleton and place into ZR BBashing lysis tube
  • Add 500 μL of DNA/RNA shield
  • Vortex for ~1 minute
  • Remove supernatant and transfer into 1.5mL tube
    • I was able to remove ~450μL

DNA Extraction

  1. Add equal volumes of DNA lysis buffer as sample volume to the 5 mL tube with sample
    • 450 μL for the fragment samples
    • Vortex to mix
  2. Transfer 700 μL into DNA filter column (yellow)
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove flow through liquid and transfer into a new 5 mL tube labeled for RNA
  3. Repeat steps 2 until all liquid is gone
  4. Add 400 μL of DNA/RNA prep buffer to column
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  5. Add 700 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  6. Add 400 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 2 minutes
    • Remove the flow through and transfer to waste
  7. Removed column and place into a new sterile 1.5 mL tube
  8. Add 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Keep flow through in tube
  9. Add another 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Discard column and keep flow through in tube
  10. After quantifying on the Qubit, I aliquoted into two 1.5 eppindorf tubes (10 μL and the remaining 89 μL) and stored at -20°C
    • Tubes were labelled: DNA #1 KW 03/13 and DNA #2 KW 03/13

RNA Extraction

  1. Add equal volumes of 100% Ethanol as sample volume to the 5 mL tube with sample
    • 900 μL for the fragment samples
    • Vortex to mix
  2. Transfer 700 μL into RNA filter column (green)
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove flow through liquid and transfer into a new 5 mL tube labeled for Protein and store in -80°C
  3. Repeat steps 12 until all liquid is gone
  4. Add 400 μL of DNA/RNA wash buffer to column
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  5. Make DNase I master mix: [75μL DNA digestion buffer and 5μL DNase] X n (sample #)
    • Two samples, 150μL DNA digestion buffer and 10μL DNase
  6. Add 80μL of DNase I master mix directly to the filter of each column. Incubate at room temperature for ~15 minutes
  7. Add 400 μL of DNA/RNA prep buffer to column
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  8. Add 700 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  9. Add 400 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 2 minutes
    • Remove the flow through and transfer to waste
  10. Removed column and place into a new sterile 1.5 mL tube
  11. Add 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Keep flow through in tube
  12. Add another 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Discard column and keep flow through in tube
  13. Aliquot into appropriate tubes for storage at -80°C

Quantification

Qubit

  • For the Qubit readings, 10 μL of each standard were used and 1 μL of each sample were used.

Table 2: Qubit readings for the extracted DNA and RNA samples.

  Standard 1 Standard 2 #1 (ng/uL) #2 (ng/uL)
DNA 199.37 21533.38 10.8 29.2
RNA 401.46 11418.56 23.6 34.2

Tapestation

  • Only RNA samples were run on the tape station

Tapestation results

Sample #1: Sample#1

Sample #2: Sample#2

Conclusions

  • Samples are good for the Illumina RNA-Seq workshop
  • Changes in Ethanol addition in RNA extraction step potentially produced higher concentrations of RNA
Written on March 13, 2019