TITLE: Protocols from: Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection
AUTHORS: Ryan  K Schott, Leah Perez, Matthew A Kwiatkowski, Vance Imhoff, Jennifer M Gumm

[DESCRIPTION]
Protocols used to extract mRNA from frog retinas, create cDNA libraries, and amplify opsins by PCR for sequencing at the UT core facility under their standard protocols. These protocols were used to obtain the opsin sequences in the paper: Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection.

[STEPS]
SECTION: RNA Extraction
1. Transfer sample into a 1.5 mLl collection tube.
SECTION: RNA Extraction
2. Pipette off RNALATER.
SECTION: RNA Extraction
3. Add 600 µL Buffer RLT.
SECTION: RNA Extraction
4. Add 6 µL Beta-mercaptoethanol.
SECTION: RNA Extraction
5. Disrupt tissue with sterile pestle.
SECTION: RNA Extraction
6. Pipette into Qiashredder column. Spin 2 min @ 8,000rpm.
SECTION: RNA Extraction
7. Remove Qiashredder column; Add cap; Spin 3 min @ max speed.
SECTION: RNA Extraction
8. Add 600 µL 70% Ethanol to new collection tube.

SECTION: RNA Extraction
9. Transfer lysate to the collection tube; mix lysate and 70% Ethanol by pipetting.
SECTION: RNA Extraction
10. Transfer lysate to RNeasy column (700 µL at a time). Spin 15 s @ 9,800rpm; Discard flow through. Add rest of lysate; Spin 15 s @ 9,800rpm; Discard flow through.
SECTION: RNA Extraction
11. Add 700 µL Buffer RWI. Spin 15 s @ 9,800rpm.
SECTION: RNA Extraction
12. Transfer RNeasy column to new collection tube.
SECTION: RNA Extraction
13. Add 500 µL Buffer RPE. Spin Spin 15 s @ 9,800rpm; Discard flow through.
SECTION: RNA Extraction
14. Add 500 µL Buffer RPE – Spin 1 min @ 9,800rpm; Discard Flow through/ Spin 2 min @ 13,000rpm.
SECTION: RNA Extraction
15. Transfer RNeasy column to new collection tube.
SECTION: RNA Extraction
16. Elute with 30 µL RNAse-Free H20. Spin 1 min @ 13,000rpm.
SECTION: RNA Extraction
17. Elute with 30 µL RNAse-Free H20- Spin 1 min @ 13,000rpm.
SECTION: CDNA Synthesis
18. Combine mRNA and RNAse-free H20 to standardize all samples to aliquots containing 0.4 µg mRNA total in 10 µL.
SECTION: CDNA Synthesis
19. Make 2 Master mixes:
SECTION: CDNA Synthesis
20. Pipette 3 µL of Master Mix 1 into each sample.
SECTION: CDNA Synthesis
21. Place sample on dry bath at 65 °C for 5 min.
SECTION: CDNA Synthesis
22. 6.5 µL Put samples on ice for 1 min.
SECTION: CDNA Synthesis
23. Pipette 6.5 µL of Master Mix 2 into each sample.
SECTION: CDNA Synthesis
24. Pipette 1 µL Superscript into each sample.
SECTION: CDNA Synthesis
25. 65 °C Incubate samples at room temp for 10 min.
SECTION: CDNA Synthesis
26. Incubate samples at 42 °C for 50 min.
SECTION: PCR
27. Keep all reagents on ice at all times.
SECTION: PCR
28. Make a master mix. Per sample add the following:
2.0 µL 10X Buffer
1.0 µL 50 millimolar (mM) MgSO4
0.5 µL dNTP mix (10 micromolar (µM) each)
18.4 µL ddH2O
1 µL forward primer (10 micromolar (µM))
1 µL reverse primer (10 micromolar (µM))
0.5 µL Taq polymerase

SECTION: PCR
29. Mix well by spinning.
SECTION: PCR
30. Add 24 µL of Master Mix to each PCR tube.
SECTION: PCR
31. Add 1 µL of sample for a total of 25 µL per tube.
SECTION: PCR
32. Program the thermocycler for the following program:
95 °C for 10 min
94 °C for 2 min
REPEAT FOLLOWING 3 steps 35-50 times:
        94 °C for 30 s
        45-50 °C for 1 min *temperature depends on primer
        72 °C for 2 min
72 °Cfor 2 min
4 °C hold
SECTION: CDNA Synthesis
19.1. Master Mix 1: add 1 µL dNTP mix and 2 µL dT primer per sample.
SECTION: CDNA Synthesis
19.2. Master Mix 2: add 4 µL Buffer, 2 µL DTT and 0.5 µL RNAase inhibitor per sample.