Protocol 41358: Probe Assay - 4933411K16Rik<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

Mut = 93 bp

Wt = 108 bp

Fam = Mut

Hex = Wt

>chr19:42041693+42041800 108bp TCGAGTCACCAGAGTCACCA CAGGGACACATGGTTTCAAG

Sequence

WT Sequence (deletions in lower case):

GGTGGTGCTGGGGACCGCTCTCTTGCTCGGAGGTGGAGGCCTCATGCTGTGGGCTCACAGGAAGAAAAGGAAGGCAGccactgagactacagaagacaagcctctggaatcccaccgagcaaatgacagctggatcaaatctcacttcagccgcctatccgaggagaggttgcccacctaccgctatgtcagcaacaatggccactcacctgaaagcaggcatggggaagtcaataccaccctccacgtggataccctcaccaccaaacacggagaaaggggcgcggctctgcaccgagattcgtttgccagcaaacagaagatatctggaagctcaatgaccaaagagatgcagagggagtcagggaagtccccgtccatggaggatgacacctgggctgctgtggcagcttgtacgaaggagattgatgccaaggggcatcgcgtggctaactccatgctgcagcgctccacaactcaccgtcgtaaaggccatgcagaatccaggaacatcagcccagaagagctgaaggctctggaggaggtggagataaaactgaaagggaatttcctaactcaccatgaaactggagtagctggggccaaccaatcgcacacagtctatagccagagtcgccatagtaaccaaagccaccattcctatccaagccaccagagcaaccagagtcatcccgtttactccagctaccagggtcatcatccaagtcacttgagcccccagagttatccaagttactcaagtcaccagagtcatccgggccactccaaccaccagggtcattcaggcctctcaagccaccagactcatctgggccactccaaccaccagggtcatccaggtcactcgagtcaccagagtcaccagggtcagccaggtcacccaagccaccagagccataatctgcccaaccgcagaaacCCAATCTATGGCTCTTGAAACCATGTGTCCCTGCCCCCCACAGGGCTGGCT

This mutation is an 863 bp deletion beginning at Chromosome 19 position 42,040,905 bp and ending after 42,041,767 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58132 CAG GGA CAC ATG GTT TCA AG Common A
58133 TCG AGT CAC CAG AGT CAC CA Wild type Forward A
58134 CTC TCT TGC TCG GAG GTG GA Mutant Forward A
58135 Fluorophore-1 CAA GCC ACC AGA GCC ATA ATC T Quencher-1 WT Probe
58136 Fluorophore-2 CAG GAA GAA AAG GAA GGC AGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58132 0.40 uM
58133 0.40 uM
58134 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.