Protocol 48436: Probe Assay - Tmem132c<em1(IMPC)J>-Alternate1
Version 2.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr5:127359407+127359488 82bp GTTGGCAGTTTCCCGAAG ACGCAGTGATTTAGGACAAA

Mut= 114 bp

Wt= 82 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

GTTCCTCTGTCTCTGCTCCCGCCCCACCCCCACCCAAGTGCTAGGACCACACCCATAAGCCACCATGCACTGCCTCTTTTGTTTTAGATTATGGAGATCTGAACTCAAGTCCTCAAGTTTTCATGGCAAGCACTTTACTAAGCCACCAACTCCCAGTTTTACACACTTCAATCTACTTTATATAATTAAACCTAATAATTCTGCAATTAGTGGACCACCGGGAAGAGTTGGCAGTTTCCCGAAGGACTCCGTAGctttggtcgtcgtggactttagaactcgtagctctttgtcctaaatcactgcgtgcaaactaacagcccggataatagctgcctgttactgatttaaacttcttcccctctcttccttagtgaccgaaagccgaggggtcatggataatatacagagattctcttcattgccgccgtacctgcccgtgagcttccacgtcctcagagccgagactgcattcttcctaaaggaggccaaccccgacccgctgcggaatgccagcctgcagtccagggtggagtctttcttcatctacaaggcccagcagcccccggtattaaacgtcagctatgggccttactctgcagaaaaggtcatccctctggacttgatgttgaaccccaactttttaggcccaaccagtaagtttccctttgactggaggctgaaggcctacatccttcaagagaaagtctacctgagccatcccaaagtacaggtgctcttccacatcgtgggccgagactgggatgaccacagggacgagaaactgccctgcctgcgggtctttgcgttcagagacagccgggaggttcgaggcagctgtcgtctgggtgggcccctgggactgtgcgtggcccagctggagatgctgcctggctggttcagtcccccagcggttgtatctgggcgcaggaggccagcagagcggccagaggggagtccggtggaactgtattatgctgtacagccaggggacgagcgtggtgactgcactggaggtgacaccaggaaggacaatgctattcgtccaggaaaggatggacaggagggcaggacatcccacctacagaagattggcaccattagcctttaccgcgcccaggacagcacccagctcagcgaactgcgcctggatggcaatgtggtcatctggctgccctcccagcccgtcaagcagggagacatagtcaccgcatctgtcaccatcgccaataactctactgtggaccatttcatcctaaggtaggcacccaggctggtatctgattgtatgtagtatgtctagggtcaatctgactcctcgcctaagggtcggcgtaattgctacctcccaagGGATCTGCCCCCTAATACAGAAACCAGTGATTCCAAGCCAAGATGCGCTGGAAAGCTGTGCTTAGGCTGTGTTTGTTGTGGGCGATGAACGGGAAGAGGCGAGTTAGTAGGTGGCCTCTTTCTCTCTATCAGCTACAGAATTAAAATTCAGGAAAACCACCGAGCAAGATCAATGCCTTTACTTGCAAATGTTATAGACTTCTCAGATAAAGAAAGGCTCCCCAGAACTGGGGAGACCACCCACTTTAAGAGGCTTGGGGTTTTTTGTTTTGTTTTGTTTTGTTTTGTTTGT

This mutation is a 1,099 bp. deletion of Chr5:127,359,435-127,360,533 (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
73352 GTT GGC AGT TTC CCG AAG Common A
73353 ACG CAG TGA TTT AGG ACA AA Wild type Reverse A
73354 ATC GCC CAC AAC AAA CAC Mutant Reverse A
73355 Fluorophore-1 CGT CGT GGA CTT TAG AAC TCG Quencher-1 WT Probe
73356 Fluorophore-2 ATT CCA AGC CAA GAT GCG CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
73352 0.40 uM
73353 0.40 uM
73354 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.