Protocol 32090: Probe Assay - Itfg2<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

Mutant=  99 bp

Wild Type = 116 bp

>chr6:128366298-128366413 116bp ATGGAGGTCACATGACAAGC CGACCCCAACACAAGTCAAC

Sequence

Wt Sequence (deleted region in lower case)

CCTGGTTGGCCTGGAATTCACACCAGTCCTTCTGCCTCGGCCTTATGAGTGCTAGAGGGGTATGGGTGTGCACCATACATTGGAAAAAATTTTCTTTTTTAATTTCTTTCCCTAAATCTGAGATTTTTACCTCCTTAAGTATCCACTTTgCTATATGGACAAACCAAACTGTACAGCTCAGTGTGTCTGTTTATTAATAAAGACAATTTATGGAGGTCACATGACAAGCTTTCATGAATGCTTTGGAAGTTTCGAGATgaatgttctctcttggtacctctctcaggttccctttctctcttgcagttgacttgtgttggggtcggagatgtctgtaataaagggaaggtaagcacgtgggtgagggtagagctccaggcccttgcctccattgtgactcccagggactcccagggaacgtcagcctctgataagctctccccaaccctcgtgtctctcctgtgcctctctgtctgtctctgtctgtctgtctgtctgtctgtctccctctctctctttctctctctctctctctcccagaacctggtggtagcagtcagcgcagaaggctggctgcacttgtttgacctgacacccaccaaggctctggatgcctctgggcaccatgagacacttggagaggagcagcgacctgtcttcaagcagcacatcccggccaacacaaaggtcatgctgatcagcgacatcggtgggcatgagcagccaaggggcgggggccagtgggttcctgggcagaccccagggGACTGGGCCAAGATGGAGAGTTGTTGCCATAGCAACCCTAGGTGTGGAGAGCTGGCACCTTGgtGCACATGTGCAGTGTTTACCTTCTCTCTTTGAATTTTGTTTTACTTTCTCCTCATTTGTTGTATGTTTAATGTATAGCTACTGAGCTCCTGTTACAAGCCAGGAACTGTTCTAGGTTCTAGAATTACAAAAATGAATGAAGCAAGCAAAAAAAAAAAAAAAAATCATGACCTCTAAGAATAGACAGAA

507 bp deletion beginning at Chromosome 6 negative strand position 128,416,346 bp and ending after 128,415,840 bp (GRCm38/mm10). In addition, there are 2 small intronic indels, a 1 bp (G) deletion 108 bp before the exon deletion and a 2 bp deletion (GT) 62 bp after the deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38329 ATG GAG GTC ACA TGA CAA GC Common A
38330 TCT CCA CAC CTA GGG TTG CT Mutant Reverse A
38331 CGA CCC CAA CAC AAG TCA AC Wild type Reverse A
38332 Fluorophore-1 TGG GCC AAG ATG GAG AGT T Quencher-1 MUT Probe
38333 Fluorophore-2 TGG TAC CTC TCT CAG GTT CCC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38329 0.40 uM
38330 0.40 uM
38331 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.