Jan 18, 2022

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

DOI
dx.doi.org/10.17504/protocols.io.b3yqqpvw
  • Ryan K Schott1,
  • Leah Perez2,
  • Matthew A Kwiatkowski2,
  • Vance Imhoff3,
  • Jennifer M Gumm3
  • 1York University;
  • 2Stephen F. Austin State University;
  • 3US Fish and Wildlife Service
  • Schott Lab
Ryan K Schott
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DOI: dx.doi.org/10.17504/protocols.io.b3yqqpvw
External link: https://doi.org/10.1002/ece3.8595
Protocol Citation: Ryan K Schott, Leah Perez, Matthew A Kwiatkowski, Vance Imhoff, Jennifer M 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.b3yqqpvw
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 18, 2022
Last Modified: January 27, 2022
Protocol Integer ID: 57072
Abstract
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.
Materials
Frog retinas, RNAEasy Kit, Qiashredder.
RNA Extraction
RNA Extraction
Transfer sample into a Amount1.5 mL l collection tube.
Pipette off RNALATER.
Add Amount600 µL Buffer RLT.
Add Amount6 µL Beta-mercaptoethanol.
Disrupt tissue with sterile pestle.
Pipette into Qiashredder column. Spin Duration00:02:00 @ 8,000rpm.
2m
Remove Qiashredder column; Add cap; Spin Duration00:03:00 @ max speed.
3m
Add Amount600 µL 70% Ethanol to new collection tube.

Transfer lysate to the collection tube; mix lysate and 70% Ethanol by pipetting.
Transfer lysate to RNeasy column (Amount700 µL at a time). Spin Duration00:00:15 @ 9,800rpm; Discard flow through. Add rest of lysate; Spin Duration00:00:15 @ 9,800rpm; Discard flow through.
30s
Add Amount700 µL Buffer RWI. Spin Duration00:00:15 @ 9,800rpm.
15s
Transfer RNeasy column to new collection tube.
Add Amount500 µL Buffer RPE. Spin Spin Duration00:00:15 @ 9,800rpm; Discard flow through.
15s
Add Amount500 µL Buffer RPE – Spin Duration00:01:00 @ 9,800rpm; Discard Flow through/ Spin Duration00:02:00 @ 13,000rpm.
3m
Transfer RNeasy column to new collection tube.
Elute with Amount30 µL RNAse-Free H20. Spin Duration00:01:00 @ 13,000rpm.
1m
Elute with Amount30 µL RNAse-Free H20- Spin Duration00:01:00 @ 13,000rpm.
1m
CDNA Synthesis
CDNA Synthesis
Combine mRNA and RNAse-free H20 to standardize all samples to aliquots containing Amount0.4 µg mRNA total in Amount10 µL .
Make 2 Master mixes:
Master Mix 1: add Amount1 µL dNTP mix and Amount2 µL dT primer per sample.
Master Mix 2: add Amount4 µL Buffer, Amount2 µL DTT and Amount0.5 µL RNAase inhibitor per sample.
Pipette Amount3 µL of Master Mix 1 into each sample.
Place sample on dry bath at Temperature65 °C for Duration00:05:00 .
5m
Amount6.5 µL Put samples on ice for Duration00:01:00 .
1m
Pipette Amount6.5 µL of Master Mix 2 into each sample.
Pipette Amount1 µL Superscript into each sample.
Temperature65 °C Incubate samples at room temp for Duration00:10:00 .
10m
Incubate samples at Temperature42 °C for Duration00:50:00 .
50m
PCR
PCR
17m 30s
17m 30s
Keep all reagents on ice at all times.
Make a master mix. Per sample add the following:
  • Amount2.0 µL 10X Buffer
  • Amount1.0 µL Concentration50 millimolar (mM) MgSO4
  • Amount0.5 µL dNTP mix (Concentration10 micromolar (µM) each)
  • Amount18.4 µL ddH2O
  • Amount1 µL forward primer (Concentration10 micromolar (µM) )
  • Amount1 µL reverse primer (Concentration10 micromolar (µM) )
  • Amount0.5 µL Taq polymerase

Mix well by spinning.
Add Amount24 µL of Master Mix to each PCR tube.
Add Amount1 µL of sample for a total of Amount25 µL per tube.
Program the thermocycler for the following program:
  • Temperature95 °C for Duration00:10:00
  • Temperature94 °C for Duration00:02:00
  • REPEAT FOLLOWING 3 steps 35-50 times:
  • Temperature94 °C for Duration00:00:30
  • Temperature45-50 °C for Duration00:01:00 *temperature depends on primer
  • Temperature72 °C for Duration00:02:00
  • Temperature72 °C for Duration00:02:00
  • Temperature4 °C hold
17m 30s