Protocol 37845: Probe Assay - Gm28729<em1(IMPC)J> Alternate 1
Version 2.0

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

>chr9:96517780-96517892 113bp TTGGCAGCAAAAATGTTGAG CAGAAACAAAGCCTGCAAGA

Mut = 114 bp

Wt = 113 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GCATCAAATCCCATTACAGATGGCTGTGAGCCACCATGTGGTTGCTGGGAATTGGTCTCAGGACCTCTGGAAAAGCAGTCAGTGCTTTTAACCTCTGAGCCATCTCTCCAACCCCTGGACTATTTAATTTAAAAgaaggtagttgagaaacttagaaacatggctaagaaactaagttccatgcctttagctctagtacttgagaggcagaggcaggtggatttctgtgagttccaggtcaaactagtctacatagtaagttccaggccacctagggctacatagtgagaccctgcctcaaccaaacaaaacacacagagaaagaaagggagggagggagggacagagggagggaccgagggaccgagggaggaagggagggagggaggaaaggaggaagggaaggagagagacagagagagagtgaataaaggacaaactagaacagagcctttagaaaagatagtgttcaatgaataattgttttaaaagaggaaaacttaattttcatgctatgtttccttgtagaaaatcatcttggcagcaaaaatgttgaggtccctgagaggctcaacttgggaacacatgggaactcagagcacactggtagccctgatggaagtttcttcatcttgcaggctttgtttctggagcagaaggggcacaccacttgcccagtgactacattggaaatcattgattgcaagatggatctgtggtctctggggcgacttgggaaagctctgcagttcagcagtttgcacttcctggtcctcgactactgcaagtaaggctgaagcagaactctgaaggctgtgcagagatggtgacagccagaggagggagggggaggggaggaccacacccgggttctctgtgcctctagccttcctcctgtacccaagtaaatgctagtgttcccttttgttctttcaaacaagtgtcttagggtttctagtgctgcaccactgggtctcgcataaattcactgtactggctggttttatggcaacttgatacaagttagagttatcagagaggaggaggctcaactgagaaaattcctccataaaaTTTTTCTGTAGGAGCTGGGATTAAAGGTGTGCACGACCCCACCGCCCAGCTCCTATAGCTGAATCTATGGAGGAATTTTCTCAATCGAGGCT

This mutation is a 940 bp deletion beginning at Chromosome 9 position 96,517,356 bp and ending after 96,518,295 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50621 TTG GCA GCA AAA ATG TTG AG Wild type Forward A
50622 CAG AAA CAA AGC CTG CAA GA Wild type Reverse A
50623 ATC TCT CCA ACC CCT GGA CT Mutant Forward A
50624 AGA AAA TTC CTC CAT AGA TTC AGC Mutant Reverse A
50625 Fluorophore-1 TGG GAA CAC ATG GGA ACT CA Quencher-1 WT Probe
52318 Fluorophore-2 CTG GGA TTA AAG GTG TGC ACG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50621 0.40 uM
50622 0.40 uM
50623 0.40 uM
50624 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.