Protocol 31715: Probe Assay - Mep1b<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

>chr18:21086048+21086146 99bp AGCGCAATTCAATGTTGTTG AACAGGGTGTTTGCCTCACT

Mutant=  103 bp

Wild Type = 99 bp

Sequence

Wt Sequence: ccagagcagccatgttgctgggttgaaattcctttacagagcgcaattcaatgttgttgtgcagtgtgtccccccactGAAAAGGGAAGGACATATAAGGTAAGGAAGTGAGCTCCACAGTGAGGCAAACACCCTGTTCCTGGTCTGTATCAGGTGCTGGTCTTCAGTGGGAAACATTCATGCTGGGAAGCAGGAGTTGTCCATTGGGACAAACTGTGACAGAATAGCAACAGTTCAGCATGAGTTCCTCCACGCCCTGGGATTCTGGCATGAGCAGTCACGTGCTGACCGGGATGATTACGTCATTATAGTTTGGGACAGGATTCAGCCAGGTATGTTTCTCTTACATGCTTAATTACAGGTCAAGTTTCTGAGCAGGTAAGCATCAGTAGCAGCCTGGTAATGGGTGTTTGGAGACCAGATACACTTCATCATGTCTACAAATTCATCTGATATCAAGGTGCAATCAGTTGTCCAATAACAGGGAGCTAAGTAGCCTGGTAtcttctctaaatactctcaacttaatgttttaaaatgattatctttggagctc

 

Deleted Region: GAAAAGGGAAGGACATATAAGGTAAGGAAGTGAGCTCCACAGTGAGGCAAACACCCTGTTCCTGGTCTGTATCAGGTGCTGGTCTTCAGTGGGAAACATTCATGCTGGGAAGCAGGAGTTGTCCATTGGGACAAACTGTGACAGAATAGCAACAGTTCAGCATGAGTTCCTCCACGCCCTGGGATTCTGGCATGAGCAGTCACGTGCTGACCGGGATGATTACGTCATTATAGTTTGGGACAGGATTCAGCCAGGTATGTTTCTCTTACATGCTTAATTACAGGTCAAGTTTCTGAGCAGGTAAGCATCAGTAGCAGCCTGGTAATGGGTGTTTGGAGACCAGATACACTTCATCATGTCTACAAATTCATCTGATATCAAGGTGCAATCAGTTGTCCAATAACAGGGAGCTAAGTAGCCTGGTA

425 bp deletion beginning at Chromosome 18 positive strand position 21,086,087 bp and ending after 21,086,511 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37463 Fluorophore-1 TGT CCC CCC ACT TCT TCT C Quencher-1 MUT Probe
37464 AGC GCA ATT CAA TGT TGT TG Common A
37465 TGA GTT ATC ATG AGC TCC AAA G Mutant Reverse A
37466 AAC AGG GTG TTT GCC TCA CT Wild type Reverse A
37467 Fluorophore-2 AGG GAA GGA CAT ATA AGG TAA GGA AG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37464 0.40 uM
37465 0.40 uM
37466 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.