Jan 18, 2022
Protocols from: Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection
- Ryan K Schott1,
- Leah Perez2,
- Matthew A Kwiatkowski2,
- Vance Imhoff3,
- Jennifer Gumm3
- 1York University;
- 2Stephen F. Austin State University;
- 3US Fish and Wildlife Service
Protocol Citation: Ryan K Schott, Leah Perez, Matthew A Kwiatkowski, Vance Imhoff, Jennifer Gumm 2022. Protocols from: Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection. protocols.io https://dx.doi.org/10.17504/protocols.io.b3wgqpbw
Manuscript citation:
Schott RK, L Perez, MA Kwiatkowski, V Imhoff, JM Gumm. 2022. Evolutionary analyses of visual opsin genes in frogs and toads: diversity, duplication, and positive selection. Ecology and Evolution (in press).
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: January 15, 2022
Last Modified: January 18, 2022
Protocol Integer ID: 57000
Abstract
Protocols used to extra mRNA from frog retina, create cDNA libraries, and prepare sampled for sequence at the UT core facility under standard protocols.
Materials
Frog retinas.
mRNA Extraction
mRNA Extraction
Transfer sample into a 1.5ml collection tube
Pipette off RNALATER
Add 600 µL Buffer RLT
Add 6 µL Beta-mercaptoethanol
Disrupt tissue with sterile pestle
Pipette into Qiashredder column- Spin 00:02:00 @ 8,000rpm
2m
Remove Qiashredder column; Add cap - Spin 00:03:00 @ max speed
3m
Add 600 µL 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 (700 µL at a time)- Spin 00:00:15 @ 9,800rpm; Discard flow through/ Add rest of lysate; Spin 00:00:15 @ 9,800rpm; Discard flow through
30s
11. Add 700 µL Buffer RWI- Spin 00:00:15 @ 9,800rpm
15s
12. Transfer RNeasy column to new collection tube
13. Add 500 µL Buffer RPE- Spin 00:00:15 @ 9,800rpm; Discard flow through
15s
14. Add 500 µL Buffer RPE – Spin 00:01:00 @ 9,800rpm; Discard Flow through/ Spin 00:02:00 @ 13,000rpm
3m
15. Transfer RNeasy column to new collection tube
16. Elute with 30 µL RNAse-Free H20- Spin 00:01:00 @ 13,00rpm
1m
17. Elute with 30 µL RNAse-Free H20- Spin 00:01:00 @ 13,00rpm
1m
cDNA Synthesis
cDNA Synthesis
1h 6m
1h 6m
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 1 µL dntp mix and 2 µL dT primer per sample
Master Mix 2: add 4 µL Buffer, 4 µL DTT and 0.5 µL RNAase inhibitor per sample
3. Pipette 3 µL of Master Mix 1 into each sample.
4. Place sample on dry bath at 65 °C for 00:05:00 .
5m
5. Put samples On ice for 00:01:00 .
1m
6. Pipette 6.5 µL of Master Mix 2 into each sample.
7. Pipette 1 µL Superscript into each sample.
8. Incubate samples Room temperature for 00:10:00 .
10m
9. Incubate samples at 42 °C for 00:50:00 .
50m
PCR
PCR
17m 30s
17m 30s
1. Keep all reagents on ice at all times.
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
3. Mix well by spinning.
4. Add 24 µL of Master Mix to each PCR tube.
5. Add 1 µL of sample for a total of 25 µL per tube.
6. Program the thermocycler for the following program:
95 °C for 00:10:00
94 °C for 00:02:00
REPEAT FOLLOWING 3 steps 35-50 times
94 °C for 00:00:30
45 °C -50 °C for 00:01:00 *temp depends on primer
72 °C for 00:02:00
END Repeat
72 °C for 00:02:00
4 °C hold
17m 30s