DNA/RNA Extraction Protocol for Coral Larvae

Protocol for Coral Larvae/Eggs DNA/RNA Extraction

Goal: Used for extraction high quality, quantity, and purity DNA and RNA from coral larvae or eggs. Can be used on samples stored in DNA/RNA Shield or flash frozen. For samples with more than 50 larvae, a fraction can be saved for the potential to do a second extraction.

Materials and Equipment

Steps

Note all steps of sample preparation, DNA, and RNA extraction are done at the RNA bench and with filter tips
QC steps can be done at the Putnam bench and don’t require filter tips

Sample preparation

Flash frozen samples in tubes

  • Try to take out samples from -80 one at a time if possible or quickly to avoid them thawing
  • Add 1mL of DNA/RNA Shield to each tube
  • Invert tube a few times, especially if larvae/eggs are frozen to the sides or lid of the tube
  • Repeat for all tubes and wait until larvae have thawed to proceed
  • If samples have 50+ larvae/eggs in each tube, you can/should save half to have the ability to repeat the extraction if necessary:
    • Label a new 1.5mL tube with the sample ID and write “saved fraction” on it to be able to identify it as already halved
    • Pipette 500ul of the sample tube into the new tube, taking the care to try to pipette half of the larvae/eggs with the liquid. It may take a few tries to get half.
    • Put the saved tubes on an ice bucket for the duration of the extraction, this allows the shield to penetrate the larvae and preserve the nucleic acids
    • When you’re done with the extraction, but the saved fraction tubes in the -80 in the original sample box
    • If you need to extract from these samples, you will not need to add any extra shield, and will be treated like a sample in shield with less than 50 larvae

Samples stored in DNA/RNA Shield

  • Thaw all samples on an ice bucket
  • Determine the volume in each tube as well as the number of larvae/eggs in each tube
    • If there are more than 50 larvae/eggs per tube, a save fraction should be made
    • If there are more than 50 larvae/eggs per tube and the volume is less than 1mL, DNA/RNA shield needs to be added to get the volume to 1mL
    • If there are less than 50 larvae/eggs per tube and the volume is less than 500ul, DNA/RNA shield needs to be added to get the volume to 500ul
    • If there are less than 50 larvae/eggs per tube and the volume is between 500ul and 1mL, no shield needs to be added
  • Add the appropriate amount of DNA/RNA Shield to the sample tubes that need it
  • Vortex and spin down briefly after adding new shield
  • Make save fractions for the samples you can, so you will have the ability to repeat the extraction if necessary:
    • Label a new 1.5mL tube with the sample ID and write “saved fraction” on it to be able to identify it as already halved
    • Pipette 500ul of the sample tube into the new tube, taking the care to try to pipette half of the larvae/eggs with the liquid. It may take a few tries to get half.
    • These saved tubes can be stored in the original sample box in the -80 immediately, or you can store on ice until after you finish the extraction and are putting everything away
    • If you need to extract from these samples, you will not need to add any extra shield, and will be treated like a sample in shield with less than 50 larvae

Homogenization

  • Spin down sample tubes to make sure there aren’t any bubbles on the lid
  • Pour in 1/2 of the beads from a glass bead tube into each of the sample tubes
  • Vortex tubes at top speed for 2 minutes
  • Tubes after vortexing are intensely bubbly
  • To tamp down bubbles, centrifuge in the mini-centrifuge for 1-2 minutes
  • Add the appropriate volume of Proteinase K and Pro K buffer (Proteinase K is stored in the -20 in the Putnam Zymo kit enzymes box)
  • For tubes with 500ul add:
    • 50ul pro k buffer
    • 25ul proteinase K
  • If you have tubes with a larger volume you can scale up the volumes (ex for 700ul, you would use 70ul buffer and 35ul pro k)
  • Vortex tubes and spin down
  • Label new 1.5mL tubes with the sample ID
  • Remove the liquid from the sample tubes into the new tubes, avoiding any debris or the glass beads note if the volume here is more than 750ul, you will need to add to a 5mL tube instead of a 1.5mL tube
  • Make note of how much volume to remove. You should be able to remove the original amount that was in the sample tube, ex if you had 500ul at the start, you should be able to get 500ul out. Some volume will stay with the beads

DNA Extraction

  • Create 1.5mL tubes of ultrapure water and 10mM Tris HCl. You will need 100ul per sample
  • Warm 10mM Tris HCl and ultrapure water in thermomixer to 70 degrees C
  • Add equal volume DNA/RNA lysis buffer to each one of your 1.5mL sample tubes. Ex. add 500ul lysis buffer to the tube if you put 500ul of sample liquid to the 1.5mL tube
  • Vortex and spin down tubes after adding the lysis buffer
  • Set up 1 collection tube with 1 yellow spin column in it per sample. Label the column with the sample IDs
  • Transfer 700ul of the liquid in the 1.5mL tube to the corresponding yellow spin column
  • Centrifuge tubes at 16,000rcf for 30 seconds
  • Label a 5mL tube for each sample
  • Pipette out the flow through in the collection tubes into the corresponding 5mL tube and put the column back into the same collection tube
  • Transfer another 700ul (or the remaining volume) of sample liquid to the corresponding yellow spin column
  • Centrifuge tubes at 16,000rcf for 30 seconds
  • Pipette out the flow through in the collection tubes into the corresponding 5mL tube
  • If there is remaining volume of lysed samples, put the yellow column back into the same collection tube and repeat the previous 3 steps
  • If all the lysed liquid has gone through the column, transfer the yellow column to a new collection tube and discard the used one
  • Get a waste beaker from above the sink, it has a pink label that says “liquid waste”
  • Add 400ul DNA/RNA prep buffer to each yellow column
  • Centrifuge columns for 30 seconds at 16,000rcf
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Add 700ul DNA/RNA wash buffer to each yellow column
  • Centrifuge columns for 30 seconds at 16,000rcf
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Add 400ul DNA/RNA wash buffer to each yellow column
  • Centrifuge columns for 2 minutes seconds at 16,000rcf
  • Label new 1.5mL tubes for the final DNA elution: use sample ID, “DNA,” the date, and your initials
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Centrifuge the columns “dry” for 1 minute
  • Transfer the yellow columns to their corresponding newly labeled 1.5mL tubes. Leave the caps open on the 1.5mL tubes. Collection tubes can be discarded
  • Add 50ul of warmed 10mM Tris HCl to each yellow column by dripping onto the white filter
  • Incubate/let tubes rest for 5 minutes at room temp
  • Centrifuge tubes for 30 seconds at 16,000 rcf
  • Add another 50ul of warmed 10mM Tris HCl to each yellow column by dripping onto the white filter
  • Incubate/let tubes rest for 5 minutes at room temp
  • Centrifuge tubes for 30 seconds at 16,000 rcf
  • Place tubes on ice bucket - this is the DNA!
  • Label enough 8-strip tubes for your samples
  • Transfer 10ul of extracted DNA to their corresponding strip tube
  • DNA tubes can be frozen at -20
  • Strip tubes can be kept on ice if QC-ing the same day, if not they also go in the -20

RNA Extraction

  • Add equal volume 100% ethanol to each one of your 5mL tubes with the flow through from the DNA columns. Ex. add 1mL ethanol to the tube if you originally had 500ul lysed sample, and added an additional 500ul of lysis buffer, meaning ~1mL of liquid came out the yellow column
  • Vortex the tubes after adding the ethanol
  • Set up 1 collection tube with 1 green spin column in it per sample. Label the column with the sample IDs
  • Transfer 700ul of the liquid in the 5mL tube to the corresponding green spin column
  • Centrifuge tubes at 16,000rcf for 30 seconds
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Transfer another 700ul of sample liquid to the corresponding green spin column
  • Centrifuge tubes at 16,000rcf for 30 seconds
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Continue in the same way for the remaining volume in the 5mL tubes
  • Once all the liquid has gone through the column, transfer the green column to a new collection tube and discard the used one
  • Add 400ul DNA/RNA wash buffer to each green column
  • Centrifuge tubes at 16,000rcf for 30 seconds
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Create the DNase mix (DNase I is stored in the -20 freezer in the Putnam Zymo kit enzymes box):
    • 75ul DNA digestion buffer * number of samples =
    • 5ul DNase I * number of samples =
  • Flick DNase mix to mix and spin down (do not vortex, DNase is a sensitive enzyme)
  • Add 80ul of DNase mix to each green spin column
  • Incubate (let sit) columns for 15 minutes
  • Add 400ul DNA/RNA prep buffer to each green column
  • Centrifuge columns for 30 seconds at 16,000rcf
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Add 700ul DNA/RNA wash buffer to each green column
  • Centrifuge columns for 30 seconds at 16,000rcf
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Add 400ul DNA/RNA wash buffer to each green column
  • Centrifuge columns for 2 minutes seconds at 16,000rcf
  • Label new RNA,” the date, and your initials
  • Pour out the flow through into the waste beaker and put the column back into the same collection tube
  • Centrifuge the columns “dry” for 1 minute
  • Transfer the green columns to their corresponding newly labeled 1.5mL tubes. Leave the caps open on the 1.5mL tubes. Collection tubes can be discarded
  • Add 50ul of warmed ultrapure water to each green column by dripping onto the white filter
  • Incubate/let tubes rest for 5 minutes at room temp
  • Centrifuge tubes for 30 seconds at 16,000 rcf
  • Add another 50ul of warmed ultrapure water to each green column by dripping onto the white filter
  • Incubate/let tubes rest for 5 minutes at room temp
  • Centrifuge tubes for 30 seconds at 16,000 rcf
  • Place tubes on ice bucket - this is the RNA!
  • Label enough 8-strip tubes for your samples
  • Transfer 7ul of extracted RNA to their corresponding strip tube
  • RNA tubes can be frozen at -80
  • Strip tubes can be kept on ice if QC-ing the same day, if not they also go in the -80

QC

Qubit: Broad Range DNA and High Sensitivity or Broad Range RNA

  • Follow Qubit protocol
  • Read samples twice and average readings
  • Make sure to record standards

DNA Gel

  • Follow Gel Protocol
  • Suggestions are to:
    • Make a 1% gel and run for 1hr at 100V
    • Use 3ul 1kb plus DNA ladder
    • Use gel green

RNA NanoDrop

RNA TapeStation

Written on March 12, 2021