Protocol 32963: Probe Assay - Prss56<em2(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

>chr1:87185123+87185226 104bp CCCAGGGTAGGAGAACATCA GCCTGTAAGTGTGGCTGTTG

Mut= 120 bp

Wt= 104 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 ATCCAGCACAGACTACATGAATGCCAAGGTTGTACCAGAAGTGCTACCCCACCCAGCAGACTCACATTGGTACAGGGGACTCCTCCTTTTGTCCATGTCCAGCCCTTATTTTTAGGTTCCATCgtccttggggtcagattctttttgaaggtccaagtggaaggggtatgccaaacttgagacacagcagggaggaggggagagtgtgggctcaggtggtcacagttcatggctgctcaaatagttccatgtgctagaaggcgggccaagtggagagtggttgaaagatgaggcccagcccccccccccaataaagcgaaagatccgcaagagggcgcagcgaggctcaaaggattgtttctcagtaggacctggacgccctagacctcaagcccccctcctccaggacccaccggagccaggtgaaactaaaagactccaagggctgaaagcgaggtgggcctcaacgtgaagataactaaaacttttgcttcatgacgccgtttctgccattctagtacaatgtggagaaagacatcaaggcgttgccaacacgacccgggctcacggccgcatagtcggtggcagcacggctccctcgggggcctggccttggctagtgaggctgcaacttggagggttgcctctgtgcgggggtgtcctggtagcagcatcctgggtgctcacggctgcacattgcttcgccgggtacgtcgtcgagattccaggcacctgctcagcagggccacccaatccgatgggagtctaagcacccatgggccaaagcagagcagagcaggtctctgctgccccctagcggaagctagctcccaacctcgggccttaaaatgccggttgcgggtgggggtgggggtgggggtgagggatcccagggtaggagaacatcacactgaattttcccactgcagggaatctgcaccttcccggtctccctctctttcccttggGTCACAACAGCCACACTTACAGGCTCACTGGATGGTGTGGGTGCATGAGGGTCTGGGCCTCTGAGGGGGCCTCACGCCACCCTCTGTCCCTGATTGGTGTCACCCA

This mutation is a 845 bp deletion beginning at Chromosome 1 position 87,184,358 bp and ending after 87,185,202 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40354 CCC AGG GTA GGA GAA CAT CA Wild type Forward A
40355 GCC TGT AAG TGT GGC TGT TG Common A
40356 GGT TGT ACC AGA AGT GCT ACC C Mutant Forward A
40357 Fluorophore-1 TTC CCA CTG CAG GGA ATC T Quencher-1 WT Probe
40358 Fluorophore-2 CCT TAT TTT TAG GTT CCA TCG TCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40354 0.40 uM
40355 0.40 uM
40356 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.