Protocol 32030: Probe Assay - Stard9<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=  95 bp

Wild Type = 98 bp

>chr2:120666704+120666801 98bp GGACTAGCCCATCAGGTTCC TGAGCCAACAAGGGTTAGGA

Sequence

Wt Sequence (deleted region in lower case)

GAGGGTGTTCGTTCATGTGTCCAGTGTCTAGGGGTAGCAGTAGGGATCTAGAAGGGACCACATTAGTAGTGTGAACTCATCTAAGCACGATGAAGTTCACTGCAGGAAAGCATATGCCTAGGTTTCCTGCTTCTCTTATTTGTCTTATGGTGATAATCTGCCCCTCGTCTGTATgtccgtcccttttgttaGTCTCTCCTGAAAGTGCCAGCAAGTAGTCTTCTATACACTGCTGTATAGGCTAGCTCGCACACACAGCCACCCTGTGTGCTCAGGGACTCCAGGTGctttgtggtaagatgagggcatgtgggcagtgtgggtcaacagtaatgctttccttttcttgtcaccaggcctctgttgggctgactccgaggatatgtgaggtatagattctctttgggtcacgtcctccccccgtgggcttcctatgtcagcttccttcagtaaagcctaacgctctggtctgtgcagggtctcttcatcagggaggatgactgtgcctctcagccttgttcccgtagcataaaagtaaggtaagagctcaagccccagaccccagatgtcccatttccatccccagctcctttcacgtcagggtcagcagaggggtggagaaaatccgcagaggacacatcatgcagtataggaaatgggacacatactaagcctttgggttgccccggactagcccatcaggttcctcctgtgcctctgttcccctcacttcctgttgtCTCACTCTTGCTTTTGGGATTCCCCTCCTAACCCTTGTTGGCTCATTCTGGAGATTTCAGACCGACTTTCCAGACTAGTGCTAGGTACAAGGTTTTGTTTTGTTTTGTTTTGTCAGACAGCATCATGCTATGTAACCTAGGCTGGCCTCAGACCCTTTATCCTGCTGTTTTACCCTCCCAAGTGTTGTAGTTATAAGCATGATCCTCTATGGCCTAGCAAAACAAAGTTTTACCAAGCTGCATTGAGAAAAATATTGATATATGATTACTAGGTTTT

 

 

453 bp deletion beginning at Chromosome 2 positive strand position 120,666,304 bp and ending after 120,666,756 bp (GRCm38/mm10). In addition there is a 17 bp deletion (GTCCGTCCCTTTTGTTA) 96 bp before the 453 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38198 TAG GCT AGC TCG CAC ACA CA Mutant Forward A
38199 TGA GCC AAC AAG GGT TAG GA Common A
38200 GGA CTA GCC CAT CAG GTT CC Wild type Forward A
38201 Fluorophore-1 CCC TGT GTG CTC AGG GAC Quencher-1 MUT Probe
38202 Fluorophore-2 TGC CTC TGT TCC CCT CAC T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38198 0.40 uM
38199 0.40 uM
38200 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.