Protocol 37285: Probe Assay - Cldn23<em1(IMPC)J>Alternate 1
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

>chr8:35825399-35825526 128bp GAAGACAGCCACCTCCCAAG CAAGTCACCCTCAGACTCCAG

Mut = 134 bp

Wt = 128 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GGCTGGGCGAGAGGGCGGAGCGGTTGGGACGCGAGAGAGGAGGGGACTTGGTGTGAGAGGCTCAGTGGGGACAGTTCCGCGCGAGAGACAAAGAGCGCCCAGAGAGCGCAGCGAGCATCGCTGCTACCCAGCCCGGCAACCTCGCAGATTCGAGGACAGGGTGCGCAGCGGAGCAGCCACAGCTGCTAGGGACCCAAGAGGCTACGCAGGATGCGGACGCCGGTggtgatgacgctgggcatggtgctcacgccctgcgggttgctgcttaatcttgtcagtacactggccccgggctggcggctggtgaagggctttctggaccagccagtggacgtggtgctgtaccaggggctgtgggacatatgtcgcgagcagagcagtcgcgaacgcgagtgcggccagcccgacgagtggaactacttccagacccagcctgtgcaggtggcccggggactcatgatcacgtcactggccactaccgccctagggctgctgctggcttccctcggtgtgcgctgttggcaagatgagccccactacgggctagcgggcctctctggcgtggtgtttttcgtcgctggcctcttcagcctcatcccggtctcctggtataaccacttcttgtcagatcccgacgtcctggccgccccgagctcgccggtcacggtgcaggtcagctacagcctggtgctgggctacctaggcagctgcctgctgcttctgggcggcttctctctggcgctcagctttgcgccctggtgtgaagagcgttgtcgccgctgtcgcaaggcgcccccagcaggcccgcgccgcagcagcatcagcaccgtctacgtggactggccggaaccagcgctcacacctgccatcaagtactacagcgacggacagcatcggcctcctcccaccgcagagcacagggacaccagcaagctcaaggttggattcccgatgccacgaccgccacccaagtcctacaccaacccgatggatgtgcttgagggagaagaaaagaagacagccacctcccaaggtggttcctcctctcgcagcactcggccctgccaaaattcgctgccctgtgactccgaCCTGTAGACAGGGTTCACCTGAGCTCTGCCTGGAGTCTGAGGGTGACTTGAACTTCTGCCAGTACATCTGCAACTAGAACCCTGGTCGCCTTCCTCTAACTGGAGGGCAAGCCTTCCTTTTCTAGGGCCAAACTCCTCTCCAAATACTAGGTTGGGTTCATTTATCTTTTAAGGAGTTTTACTTTTCTCTCCAGAG

This mutation is a 860 bp deletion beginning at Chromosome 8 position 35,825,454 bp and ending after 35,826,313 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49325 CAA GTC ACC CTC AGA CTC CAG Common A
49326 GAA GAC AGC CAC CTC CCA AG Wild type Forward A
49327 CTC GCA GAT TCG AGG ACA G Mutant Forward A
49328 Fluorophore-1 CTC GCA GCA CTC GGC C Quencher-1 WT Probe
50972 Fluorophore-2 AGG CTA CGC AGG ATG CGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49325 0.40 uM
49326 0.40 uM
49327 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.