Sep 11, 2024

Public workspaceCHCHD2 T61I Mouse Genotyping

DOI
dx.doi.org/10.17504/protocols.io.kxygxyejwl8j/v1
  • 1Gladstone Institutes
Haru Yamamoto
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DOI: dx.doi.org/10.17504/protocols.io.kxygxyejwl8j/v1
Protocol Citation: Szu-Chi Liao, Ken Nakamura 2024. CHCHD2 T61I Mouse Genotyping. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxyejwl8j/v1
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: April 19, 2024
Last Modified: September 13, 2024
Protocol Integer ID: 98456
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: 020529
Disclaimer
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Abstract
To genotype CHCHD2 T61I point mutant mice by qPCR.
Mouse Tail Lysis
Mouse Tail Lysis
Materials
  • Isoflurane
  • Sanitized scissors
  • DirectPCR lysis reagent (mouse tail, Viagen Biotech, 102-T)
  • Proteinase K – Recombinant, PCR grade (Roche, 03115844001)
  • Microcentrifuge tubes
  • Incubator
  • Water bath
Anesthetize mice with isoflurane.
Cut no longer than 2 mm of distal tail from mice.
Place each tail in a sterile, labeled tube.
Add 200 μl of DirectPCR lysis reagent.
Add 10 μl of proteinase K and mix well.
Incubate the tails at 55°C overnight.
To deactivate proteinase K, incubate tails for 45 min in an 85°C water bath.
Store extracted DNA at -20°C until use.
qPCR Genotyping
qPCR Genotyping
Materials
  • Kapa Probe Fast qPCR Master Mix (2X) kit (Kapa Biosystems, KK4715)
  • EndPoint-F Primer: TGAAGATGGCCCAGATCTG
  • EndPoint-R Primer: CTGAAGCCCCCAGTGAT
  • WT-Probe: (5’ JOE) TTAGATGGCTACCACCGCG (3’ Iowa Black)
  • MT-Probe: (5’ 6-FAM) TTAGATGGCGATCACCGCG (3’ Iowa Black)
  • Nuclease-free water
Make Reaction Buffer:
ABC
Reagent Volume (μl) Final Concentration
qPCR Master Mix (2X) 10 1X
High ROX (50X) 0.4 1X
EndPoint-F (10 μM) 0.8 0.4 μM
EndPoint-R (10 μM) 0.8 0.4 μM
WT-Probe (10 μM) 0.4 0.2 μM
MT-Probe (10 μM) 0.4 0.2 μM
DNA 100 ng
Nuclease-free water Make up to 20 μl
Total 20 μl
Open SDS 2.4 software, create a new experiment and choose “standard curve”.
Load the plate.
Set thermal cycling as follows:
Step Temperature (°C) Duration
1 95 3 min
2 95 10 s
3 60 30 s
4 Repeat steps 2-3 for 40 cycles
5 4 ∞
In the Setup tab, add detectors for FAM and JOE signal and start the program.
After amplification is done, create a new experiment and choose “allelic discrimination”.
Assign the markers which detect FAM and JOE signals.
Start “Post Read”.
Expected Results:
When plotted WT against MT fluorescence signal, each genotype would show a distinct ratio/slope as below: