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 Gumm

[DESCRIPTION]
Protocols used to extra mRNA from frog retina, create cDNA libraries, and prepare sampled for sequence at the UT core facility under standard protocols.

[STEPS]
SECTION: mRNA Extraction
1. Transfer sample into a 1.5ml collection tube
SECTION: mRNA Extraction
2. Pipette off RNALATER
SECTION: mRNA Extraction
3. Add 600 µL Buffer RLT

SECTION: mRNA Extraction
4. Add 6 µL Beta-mercaptoethanol

SECTION: mRNA Extraction
5. Disrupt tissue with sterile pestle
SECTION: mRNA Extraction
6. Pipette into Qiashredder column- Spin 2 min @ 8,000rpm

SECTION: mRNA Extraction
7. Remove Qiashredder column; Add cap - Spin 3 min @ max speed

SECTION: mRNA Extraction
8. Add 600 µL 70% Ethanol to new collection tube

SECTION: mRNA Extraction
9. Transfer lysate to the collection tube- mix lysate and 70% Ethanol by pipetting
SECTION: mRNA Extraction
10. 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: mRNA Extraction
11. 11.  Add 700 µL Buffer RWI- Spin 15 s @ 9,800rpm

SECTION: mRNA Extraction
12. 12.  Transfer RNeasy column to new collection tube
SECTION: mRNA Extraction
13. 13.  Add 500 µL Buffer RPE- Spin 15 s @ 9,800rpm; Discard flow through

SECTION: mRNA Extraction
14. 14.  Add 500 µLBuffer RPE – Spin 1 min @ 9,800rpm; Discard Flow through/ Spin 2 min @ 13,000rpm

SECTION: mRNA Extraction
15. 15.  Transfer RNeasy column to new collection tube
SECTION: mRNA Extraction
16. 16.  Elute with 30 µL RNAse-Free H20- Spin 1 min @ 13,00rpm

SECTION: mRNA Extraction
17. 17.  Elute with 30 µL RNAse-Free H20- Spin 1 min @ 13,00rpm

SECTION: cDNA Synthesis
18. 1. Combine mRNA and RNAse-free H20 to standardize all samples to aliquots containing 0.4μg mRNA total in 10μl.
SECTION: cDNA Synthesis
19. 2. Make 2 Master mixes
SECTION: cDNA Synthesis
20. Master Mix 1: add 1 µL dntp mix and 2 µL dT primer per sample

SECTION: cDNA Synthesis
21. Master Mix 2: add 4 µL Buffer, 4 µL DTT and 0.5 µL RNAase inhibitor per sample


SECTION: cDNA Synthesis
22. 3. Pipette 3 µL of Master Mix 1 into each sample.

SECTION: cDNA Synthesis
23. 4. Place sample on dry bath at 65 °C for 5 min.

SECTION: cDNA Synthesis
24. 5. Put samples on ice for 1 min.

SECTION: cDNA Synthesis
25. 6. Pipette 6.5 µLof Master Mix 2 into each sample.

SECTION: cDNA Synthesis
26. 7. Pipette 1 µLSuperscript into each sample.

SECTION: cDNA Synthesis
27. 8. Incubate samples 65 Room temperature for 10 min.

SECTION: cDNA Synthesis
28. 9. Incubate samples at 42 °Cfor 50 min.

SECTION: PCR
29. 1. Keep all reagents on ice at all times.
SECTION: PCR
30. 2. Make a master mix. Per sample add the following:
2.0 µL 10X Buffer
1.0 µL 50mM MgSO4
0.5 µL dNTP mix (10mM each)
18.4 µL ddH2O
1 µL forward primer (10μM)
1 µL reverse primer (10μM)
0.5 µL taq polymerase

SECTION: PCR
31. 3. Mix well by spinning.
SECTION: PCR
32. 4. Add 24 µL of Master Mix to each PCR tube.

SECTION: PCR
33. 5. Add 1 µL of sample for a total of 25 µL per tube.

SECTION: PCR
34. 6. 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 °Cfor 30 s
45 °C -50 °Cfor 1 min *temp depends on primer
72 °C for 2 min
END Repeat
72 °C for 2 min
4 °C hold