Larvae from Biomineralization Project

Posted orginally by Maggie Schedl

Materials:

• Using Zymo Duet DNA/RNA Extraction Kit HERE**
• Thermoixer
• 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

Sample Preparation

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

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 Thermoixer 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 steps 18-20 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 steps 25-27 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

1. Using Broad Range dsDNA and Broad Range RNA kits
2. 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%)
3. 10µl of each standard was used to create the standard curve and 1µl of each sample was used in quantification
4. All quantifications are in ng/µl
5. All quantifications were taken twice

Tape Station

1. Follow RNA Tape Station protocol here (write protocol)
2. Only analyzing sample 398 because it is the only one with RNA

Results

2-25-19 More Test Extractions

Samples

Followed same extraction protocol as above

Quibit

Tape Station Results

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")