Protocols from: Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection

Transfer sample into a 1.5ml collection tube

Pipette off RNALATER

Add 600µL Buffer RLT

Add 6µL Beta-mercaptoethanol

Disrupt tissue with sterile pestle

Pipette into Qiashredder column- Spin 0h 2m 0s @ 8,000rpm

Remove Qiashredder column; Add cap - Spin 0h 3m 0s @ max speed

Add 600µL 70% Ethanol to new collection tube

Transfer lysate to the collection tube- mix lysate and 70% Ethanol by pipetting

10. Transfer lysate to RNeasy column (700µL at a time)- Spin 0h 0m 15s @ 9,800rpm; Discard flow through/ Add rest of lysate; Spin 0h 0m 15s @ 9,800rpm; Discard flow through

11. Add 700µL Buffer RWI- Spin 0h 0m 15s @ 9,800rpm

12. Transfer RNeasy column to new collection tube

13. Add 500µL Buffer RPE- Spin 0h 0m 15s @ 9,800rpm; Discard flow through

14. Add 500µLBuffer RPE – Spin 0h 1m 0s @ 9,800rpm; Discard Flow through/ Spin 0h 2m 0s @ 13,000rpm

15. Transfer RNeasy column to new collection tube

16. Elute with 30µL RNAse-Free H20- Spin 0h 1m 0s @ 13,00rpm

17. Elute with 30µL RNAse-Free H20- Spin 0h 1m 0s @ 13,00rpm

1. Combine mRNA and RNAse-free H20 to standardize all samples to aliquots containing 0.4μg mRNA total in 10μl.

2. Make 2 Master mixes

Master Mix 1: add 1µL dntp mix and 2µL dT primer per sample

Master Mix 2: add 4µL Buffer, 4µL DTT and 0.5µL RNAase inhibitor per sample

3. Pipette 3µL of Master Mix 1 into each sample.

4. Place sample on dry bath at 65°C for 0h 5m 0s.

5. Put samples 65On ice for 0h 1m 0s.

6. Pipette 6.5µLof Master Mix 2 into each sample.

7. Pipette 1µLSuperscript into each sample.

8. Incubate samples 65Room temperature for 0h 10m 0s.

9. Incubate samples at 42°Cfor 0h 50m 0s.

1. Keep all reagents on ice at all times.

2. Make a master mix. Per sample add the following:

2.0µL 10X Buffer

1.0µL 50mM MgSO4

0.5µL dNTP mix (10mM each)

18.4µL ddH2O

1µL forward primer (10μM)

1µL reverse primer (10μM)

0.5µL taq polymerase

3. Mix well by spinning.

4. Add 24µL of Master Mix to each PCR tube.

5. Add 1µL of sample for a total of 25µL per tube.

6. Program the thermocycler for the following program:

95°C for 0h 10m 0s

94°C for 0h 2m 0s

REPEAT FOLLOWING 3 steps 35-50 times

94°Cfor 0h 0m 30s

45°C -50°Cfor 0h 1m 0s *temp depends on primer

72°C for 0h 2m 0s

END Repeat

72°C for 0h 2m 0s

4°C hold