Protocol 33232: Probe Assay - St7<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

>chr6:17834879+17834966 88bp AGCTGTACCAGAGAAGTATCACG GATAAAGCTAATGGGAACGAAAG

Mut= 83 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TTTCAGCTAAGATGTTTTTGTTATTTGAGTATTATATTAACTTTGAGAAAAATAATTTGGAATGCACCTTAACAATATTCTTTTATCTATCACTATATTTATTGATTCTTTTAAAAACCACTCTTCCAGCAGAGAAAAACCATTTTAActaaataatattggtatttagttactaaaatctattacagttagaccaatttaaacatttttcaaaggcggatagaaaaagtaaacatatattaagggaatgtatcctgaaattacaagtagaaactgaggtcataaacacacattatctttctgttctagatatttgaatggtggtattttcgcaagtatggaacatctttcattgaacaagtctccgtaagccacttgcgcccccttctgggaggggttgacaacaactcttccaataattctaattccagtaatggagactcagattccaacagacaaagtgtctcaggtatggaacgtctcttacaaaattttcgtgaaattaacaaaactgttttaaaagattacgtttcagagaaaaactgtgtttaaatttcaagggaggtaagaaagctgtaccagagaagtatcacgcatgcatcccagggacagaaaCTATTCATGCTGTTAACTTTACTTTCGTTCCCATTAGCTTTATCATAACAAATGATAATAGCATTAGTTTTAAAGCAAATGTAGCATAGAGTATAGAGTATTACATAGAGTATCAAGTGCATTAGCTACCT

This mutation is a 467 bp deletion beginning at Chromosome 6 position 17,834,456 bp and ending after 17,834,922 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40946 AGC TGT ACC AGA GAA GTA TCA CG Wild type Forward A
40947 GAT AAA GCT AAT GGG AAC GAA AG Common A
40948 TTT AAA AAC CAC TCT TCC AGC AG Mutant Forward A
40949 Fluorophore-1 ATG CAT CCC AGG GAC AGA Quencher-1 WT Probe
40950 Fluorophore-2 AAC CAT TTT AAC TAT TCA TGC TGT TAA CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40946 0.40 uM
40947 0.40 uM
40948 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.