Testing Number of Montipora Larvae Needed for DNA/RNA Extraction

Larvae from Biomineralization Project

Using Zymo Duet DNA/RNA Extraction Kit HERE Other components: Thermoixer and centrifuge capable of 16,000 rcf spin (Eppendorf)

Make sure ethanol has been added to the wash buffer, and that enzymes have been re-hydrated before starting

  1. Take sample tube with larvae 1 at a time out of the -80 to minimize amount of thawing
  2. Add 300µl DNA/RNA shield directly to the sample tube
  3. Record tube number

Samples

Sample # Date Collected Type How Many Extracter
395 6/26/2018 larvae 15 Maggie
386 6/26/2018 larvae 3 Maggie
389 6/26/2018 larvae 5 Erin
398 6/26/2018 larvae 50 Erin
  1. Add 30µl of PK digestion buffer to each sample tube
  2. Add 15µl Proteinase K to each sample tube
  3. Vortex and spin down sample tubes
  4. Place in Thermomixer for 1 hour at 55 degrees C, shaking at 1100 rpm
  5. After digestion proceed with DNA and RNA Extraction

    DNA Extraction

  6. Set up yellow DNA spin columns and collection tubes, label appropriately
  7. Warm elution liquids to 70 degrees C (10mM Tris HCl pH. 8.0 and RNase free water)
  8. Add equal volume (345µl) DNA/RNA lysis buffer to each sample tube
  9. Finger flick to mix tubes
  10. Add 700µl (total volume) of sample gently to the yellow DNA spin column
  11. Centrifuge at 16,000 rcf (g) for 30 seconds
  12. Important Save the flow through from this step: transfer to a new 1.5mL tube labeled for RNA
  13. Add 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
  14. Centrifuge at 16,000 rcf (g) for 30 seconds
  15. Discard flow through (Zymo kit waste)
  16. Add 700µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
  17. Centrifuge at 16,000 rcf (g) for 30 seconds
  18. Discard flow through (Zymo kit waste)
  19. Add 400µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
  20. Centrifuge at 16,000 rcf (g) for 2 minutes
  21. Discard flow through (Zymo kit waste)
  22. Transfer yellow columns to new 1.5mL microcentrifuge tubes
  23. Add 50µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filer
  24. Incubate at room temp for 5 minutes
  25. Centrifuge at 16,000 rcf (g) for 30 seconds
  26. Repeat last three steps for a final elution volume of 100µl
  27. Label tubes, store at 4 degrees C if quantifying the same day or the next, if waiting longer store in -20

RNA Extraction

  1. Add equal volume (700µl) 100% EtOH to the 1.5mL tubes labeled for RNA containing the original yellow column flow through
  2. Vortex and spin down to mix
  3. Add 700µl of that liquid to the green RNA spin columns
  4. Centrifuge at 16,000 rcf (g) for 30 seconds
  5. Discard flow through (Zymo kit waste)
  6. Add 700µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
  7. Centrifuge at 16,000 rcf (g) for 30 seconds
  8. Discard flow through (Zymo kit waste)
  9. Add 400µl DNA/RNA Wash Buffer gently to each green RNA column
  10. Centrifuge at 16,000 rcf (g) for 30 seconds
  11. Discard flow through (Zymo kit waste)
  12. Make DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
  13. Add 80µl DNase I treatment master mix directly to the filter of the green RNA columns
  14. Incubate at room temp for 15 minutes
  15. Add 400µl DNA/RNA Prep Buffer gently to each column
  16. Centrifuge at 16,000 rcf (g) for 30 seconds
  17. Discard flow through (Zymo kit waste)
  18. Add 700µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
  19. Centrifuge at 16,000 rcf (g) for 30 seconds
  20. Discard flow through (Zymo kit waste)
  21. Add 400µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
  22. Centrifuge at 16,000 rcf (g) for 2 minutes
  23. Discard flow through (Zymo kit waste)
  24. Transfer green columns to new 1.5mL microcentrifuge tubes
  25. Add 50µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
  26. Incubate at room temp for 5 minutes
  27. Centrifuge at 16,000 rcf (g) for 30 seconds
  28. Repeat last three steps for a final elution volume of 100µl
  29. Label 1.5mL tubes on ice afterwards, and aliquot 5µl into PCR strip tubes to save for Qubit and Tape Station to avoid freeze-thaw of your stock sample
  30. Store all tubes in the -80

Qubit

  • Using Broad Range dsDNA and Broad Range RNA kits
  • A working stock solution was made of 199µl * n buffer + n µl Quant-IT reagent
    • n is # of samples + 2 + %error (usually % error was less than 1%)
  • 10µl of each standard was used to create the standard curve and 1µl of each sample was used in quantification
  • All quantifications are in ng/µl
  • All quantifications were taken twice
Sample DNA Standard 1 (RFU) DNA Standard 2 (RFU) DNA 1 (ng/µl) DNA 2 (ng/µl) Average DNA RNA Standard 1 (RFU) RNA Standard 2 (RFU) RNA 1 (ng/µl) RNA 2 (ng/ul) Average RNA
395 198.9 21557 3.04 3.04 3.04 386 11014 too low - -
386 198.9 21557 too low - - 386 11014 too low - -
389 198.9 21557 too low - - 386 11014 too low - -
398 198.9 21557 9.78 9.70 9.74 386 11014 15.2 14.4 14.8

Tape Station

  • Follow RNA Tape Station protocol HERE
  • Only analyzing sample 398 because it is the only one with RNA

Results

Tape1

2-25-19 More Test Extractions

Samples

Sample # Date Collected Type How Many
404 6/26/2018 larvae 100
401 6/26/2018 larvae 30
119 6/13/2018 eggs 20
102 6/13/2018 bundles 20
392 6/26/2018 larvae 10

Followed same extraction protocol as above

Quibit

Sample DNA Standard 1 (RFU) DNA Standard 2 (RFU) DNA 1 (ng/µl) DNA 2 (ng/µl) Average DNA RNA Standard 1 (RFU) RNA Standard 2 (RFU) RNA 1 (ng/µl) RNA 2 (ng/ul) Average RNA
404 196 21470 12.9 12.7 12.8 393 10798 46 45.8 45.9
401 196 21470 21 21.4 21.2 393 10798 27.8 27.6 27.7
119 196 21470 too low - - 393 10798 101 101 101
102 196 21470 70.6 71.6 71.1 393 10798 too low - -
392 196 21470 4.32 4.28 4.3 393 10798 too low - -

Tape Station Results

Tape2 Tape3 Tape4

Graph of DNA and RNA together by number of larvae

R code for the graph

QubitValues <- read.csv("larvae.csv", header = TRUE)
library(ggplot2)
ggplot()+
	  geom_bar(data=QubitValues, aes(x=Number, y=ng, fill=Type, width=8), stat = "identity", position= "dodge") +
	  theme_minimal() + ylab("ng/ul") + xlab("Number of Larvae")

graph1

Written on February 22, 2019