Protocol 36173: Probe Assay - Pfas<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

>chr11:69001876-69001977 102bp TTGAAAGCAGGTGTCCTTGG TGGCAAGAATCAGTTGGGAAG

Mut= 117 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CGCATCTCAGTAAGCACTGTGCCATTGAACAGCTTTTCTTTTGTAACATGATGTGGCTAAGTTTCCCATACTGGGTTTCTGTATGGGTCTATGTACCTGAGTGCAGCGCCAATGGAGGCATCGGGGCCCCCTAGAACTAGAGTCTCAGGCAGGTGTGAGCCTGACATGGGTACAAGGAACCAAACTTGGAATTATTTCTCCATCTTCTGATTAATTTTTGAAAGCAGGTGTCCTTGGCATCCAGTCTGAAACCAGAGGCCAGTGgtgtgttgggtcgatagcagtgggcaagatctaccttcccaactgattcttgccaaggccttcagattataaccatctgactctttcctcaggctgaatttctcgactccagcatcgaccaacattgtttctgtgtgccaggcagccgggctgagggctgtggatcgggtagagaccacccgacgttaccgactttcggtaaggtgtgggcattgtggtaggagatcaagaggagcgagtgggccagagattagatcctgaactcgagcagagtcatgggagaagcagggggaaagctcttggcttttggcctgtcctgtatgcttaggaggtttggttgtttgctgcttgaaggtacctgtgaagggagaatcggccagcctgactaaccagacttatctgtgcatccttttagtctgaacaaagacgctGCATGGGTCTGAGTGTGCATAGCTCAGTGGTGGGAATTCACCTCCTATATACAAACCCTGGCCTTGATCTCTGCACTAGAACAGAAAAGTGGTGCAATGTGGTAGTAGTGAGGTGAATAGCTTGGAGGTCCAGAGCGAGTCCTTTTTCTCTTTTCTTCAGCCTTGTCTTTTGTTTTCTTATGACTGGATCTTCCCAACACCAGTTTACCGACCACCCTACAGCTGAAATGGAAGCCATCTCTCTGGCCGCCTTGCACGACCGGATGACAGAGCAGCACTACCCTGACCCCATCCAGAGCTTCT

This mutation is a 431 bp deletion beginning at Chromosome 11 position 69,001,500 bp and ending after 69,001,930 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48017 TTG AAA GCA GGT GTC CTT GG Common A
48018 TGG CAA GAA TCA GTT GGG AAG Wild type Reverse A
48019 AGA TCA AGG CCA GGG TTT GT Mutant Reverse A
48020 Fluorophore-1 CGA TAG CAG TGG GCA AGA TC Quencher-1 WT Probe
48021 Fluorophore-2 CAT AGC TCA GTG GTG GGA ATT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48017 0.40 uM
48018 0.40 uM
48019 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.