Protocol 32629: Probe Assay - Fam24b<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

>chr7:131326222-131326315 94bp GGAGGCAGATGATGAATGCT GGAATGACAAGCCTCAGCAG

Mut= 86 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 

GGAGGACAGCTGACAGGGATGTAGGTCAGACAAGCGATGGTTTGAAGTTTGGGAGAGAAGCTTTAAGTATGAGAAAAACTGGCTGTCATGATGTCCCTTAAGAGGGACTTAGATTGTAGGGGCCAGAGCTGAGCCCAGCCAACTGCTGTGCCTTCTCTATTATCctcctgggcctgcaggagcccctggggcacagtcactctctttacttgtgatggaatctgggtttcacatgcatagcaattgtaggagaggaaaagatgggaaaggagcagagagctttaacccagggctggtgtcacatgtatgatgggttccaaccctggcctttctcacaaagccctcataattgtaagaagctcactgagaagggaaattctaagcttgttttgtgtcttttcttaagaatcgcaaaggaggcagatgatgaatgctatattgatccatgcaaggacccccatgagagcatcatcctggccaacagcatccctgctgaggcttgtcattcctaccaggccaacaccattgctgttgcgagttgcggtcccctccagtgttgtaatgtatgtggtgtctatgcagatgttaactccctgccaccctgcctttgtagcataagggaaggactctgacatggcacctatggataaaaggaaatcttcatgatcagtatttcaatttcttactagaatattttcttactcaagccaatttctatagtgtttatgttgtatactatctaacattgtgtttatgtacttttgtataAATgtacaatagtgaaaacaatctctgcctctattatgtttcctagtTATATTTCTTATACCACTATATGTATTACCACCGAGGACCAACCTCAGTCATTTTGGAGTTCTGAAGAATGGGAGGATTGGATTGGCACAAAATCAGAGACTCAGGCACAAATGCAGGTGCAAGCTGACAGGTCACCATTGCTGTT

 

This mutation is a 648 bp deletion beginning at Chromosome 7 position 131,325,916 bp for 46 bp to 131,325,962 bp retaining 3 bp TTA and then an additional 602 bp deletion ending after 131326566 (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39592 GGA GGC AGA TGA TGA ATG CT Wild type Forward A
39593 GGA ATG ACA AGC CTC AGC AG Wild type Reverse A
39594 CCA ACT GCT GTG CCT TCT CT Mutant Forward A
39595 CCA AAA TGA CTG AGG TTG GTC Mutant Reverse A
39596 Fluorophore-1 TGA TCC ATG CAA GGA CCC Quencher-1 WT Probe
39597 Fluorophore-2 TCT TAT ACC ACT ATA TGT ATT ACC ACC GAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39592 0.40 uM
39593 0.40 uM
39594 0.40 uM
39595 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.