Protocol 37964: Probe Assay - Il3ra<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

>chr14:14348981+14349065 85bp CCATGACACCTGGGTTGG CAGAGGTCACACCTGGCATC

Mut= 85 bp

Wt= 85 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GTTAGGCAAATTTATGAAGGCTAGAACATGATGTCATCTGGGTTTATGCAAATAAACTCAGATGGAACCATGATGTTATGCAAATCCATGCAGATTTATGCAAAGCCCATGATCAGGGCCTGTGGATATTTATTCAAATGAAATTCAAATTTATGCAAGTTCATACAAATTAATGCAAATTAGTGCTGGCCCTACCACTCACCCCAAgtatgggtgacccctgctgacgccgacatgcaggttgaccccaggtgaccccgcccctcacactgaccctgccctgccccctgacctctgtgaccccgcaggctgaccccgggcttgcccgctgctcattccagtcgctctccctctgccatgtgaccaacttcaccgtcttcctggggaaggacagggctgtggcaggatccatccagttccccccagatgatggtgggtgaggccccgcctccgtgacccccacgtgacccatgacacctgggttggcccccccaagaaccaactgtgacccctaaccctagctgtgaccctatgatgccaggtgtgacctctgatcccatgtgtgaccaggggtcctaccctggccccagcccatgacccttgaccccagggtgcgacccctgctcccaaATCTTACACCTGACCCCAACATGGGACCCCAACCCCGTGTGTGACCCCTGACCCTGTCCCCTCCATCATGACCGCAGTGACCCCATCCCAACCCCCACAGATGGCGACCACGAGGCCGCAGCCCAGGACCTGCGCTGCTGGGTGCATGAGGGGCAGCTGAGCTGCCAATGGGAGCGGGGTCCGAAAGCAACGGGTGACGTGCACTACC

This mutation is a 423 bp deletion beginning at Chromosome 14 position 14,348,720 bp and ending after 14,349,142 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52639 CCA TGA CAC CTG GGT TGG Wild type Forward A
52640 CAG AGG TCA CAC CTG GCA TC Wild type Reverse A
52641 AAT TAG TGC TGG CCC TAC CA Mutant Forward A
52642 AGG GTC AGG GGT CAC ACA Mutant Reverse A
52643 Fluorophore-1 AAC TGT GAC CCC TAA CCC TAG C Quencher-1 WT Probe
52644 Fluorophore-2 CAC CCC AAA TCT TAC ACC TGA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52639 0.40 uM
52640 0.40 uM
52641 0.40 uM
52642 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.