Protocol 36498: Probe Assay - Slc4a1ap<em1(IMPC)J>
Version 1.0

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

>chr5:31532176+31532297 122bp GTTAACATATGGTTGCAGAGAATAGCC CCGCTTTCATTCATTTCACC

Mut = 137 bp

Wt = 122 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 ACATACACACACACTCACAATTGATAAATAAAAGTATGTATGGAGAGATGATTGGTTTGGATGTTTTGTTCTGATTGTGCTGAGATCAAATCCAGGACCTTGCTCATGCTAGGCAAGCATTCTGTCACTAAGCAACAACCTGTGACCCCCATCTCCTGGAGACCAGGTAGGCCTTGTAGGTATGAGCTTCTTGCCTCAGTTTCTGAATGCCTAGTATTATAAGCTGTATGTATAAGTATACACAGCCAAGATTGATTTCCAGGAGGTGATccctggcagtgctcgctgtgatgttagacactgagatctttgatctgatacagtccttactggatcaagtagtaattttagacctgactgagactgttcttttcctgcaggagaagagttagaatatgaatttgatgagcaaggacacagcacttggctctgcagggtgaggtatgttcttctcgtttgtattctagctgtgatttgagaccgggtcttgttttgtggcagtcttccctcttttgcctactgagtactgggttcatgtgctctttccttgctaatgtgtatgtttgaaagtagccagtttcaagttaacatatggttgcagagaatagcccaagagtaacttcactctgcactttaggctagttccaagtgtgggccttggTAGCTTAATAGCACTGGGAGCTGGGGTGAAATGAATGAAAGCGGGTGTAAATGACTTGAAGTCCAACTGAGGCAAGAAAGAGTGAAGTTACCTGAAAGCATGAAATGCAGTCATCATGGACACTCTTGATAGCTGTTTTCATGAGAGTCAGAGCATCCTGCTGAGCTCCACCCCCTGTAAACCAATGCAGGTTACCTGTAGATGATTCAACTGGGAAACAGCTGGTGGCTGAGGCCATTCACTCAGGAAAGAAAAA

This mutation is a 391 bp deletion beginning at Chromosome 5 position 31,531,863 bp and ending after 31,532,253 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48916 CCG CTT TCA TTC ATT TCA CC Common A
48917 GTT AAC ATA TGG TTG CAG AGA ATA GCC Wild type Forward A
48918 GGT ATG AGC TTC TTG CCT CA Mutant Forward A
48919 Fluorophore-1 CTC TGC ACT TTA GGC TAG TTC CA Quencher-1 WT Probe
48920 Fluorophore-2 CAG GAG GTG ATT TAG CTT AAT AGC AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48916 0.40 uM
48917 0.40 uM
48918 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.