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

Transfer sample into a 1.5mLl 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.

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.

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

Transfer RNeasy column to new collection tube.

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

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

Transfer RNeasy column to new collection tube.

Elute with 30µL RNAse-Free H₂0. Spin 0h 1m 0s @ 13,000rpm.

Elute with 30µL RNAse-Free H₂0- Spin 0h 1m 0s @ 13,000rpm.

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

Make 2 Master mixes:

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

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

Pipette 3µL of Master Mix 1 into each sample.

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

6.5µL Put samples on ice for 0h 1m 0s.

Pipette 6.5µL of Master Mix 2 into each sample.

Pipette 1µL Superscript into each sample.

65°C Incubate samples at room temp for 0h 10m 0s.

Incubate samples at 42°C for 0h 50m 0s.

Keep all reagents on ice at all times.

Make a master mix. Per sample add the following:

• 2.0µL 10X Buffer
• 1.0µL 50millimolar (mM) MgSO₄
• 0.5µL dNTP mix (10micromolar (µM) each)
• 18.4µL ddH₂O
• 1µL forward primer (10micromolar (µM))
• 1µL reverse primer (10micromolar (µM))
• 0.5µL Taq polymerase

Mix well by spinning.

Add 24µL of Master Mix to each PCR tube.

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

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°C for 0h 0m 30s
• 45-50°C for 0h 1m 0s *temperature depends on primer
• 72°C for 0h 2m 0s
• 72°Cfor 0h 2m 0s
• 4°C hold