Protocol 36525: Probe Assay - Plb1<em1(IMPC)J> Alternate2
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

Mutant=  86 bp

Wild Type = 91 bp

>chr5:32269662+32269752 91bp CAGAGGGTGGGGAAGCAG ACGAGGCAGGGAAGCTACG

Sequence

Wt Sequence (deletion in lower case):

 ATGACAGATGGTTTTACTTTATAAATCAGTCTTCTGATTCTGTGTAGAATATAGCGTGACTTACATTATGTTTAAATGGATGCCCTGAAGAAAGCTGTGAGCCACTCTGTTATGATGGTCTAGCAGACAACCTGTGGGGAAGCAGAGGGTGGGGAAGCAGAGggtgcttgcctcggcataaagtgagcaaggtggacttgctcttctatagacccgtagcttccctgcctcgtgctggtttctcctgactgacttactcacaccaagtatctatcttttccagcctctagcccctgggtctggcacgatcaacatggagaaacctcaaaggtacccatcctggtacacagctagcagagacacgccacacactccctctgctaactccttgcttgattgatgggggacaccaaggaccagagagttcaaagtctttgcttaagggttgggggacagcagtgaccagaaccacttcctgccccctcatcctcccagaacccatgacacccgtgactcctctgggcgaatgagagagaagtgggaaagaccagagagatggaatccctgtgactgcacagggcctcctccctctgctcctgggggccttgcagggtttggactggagtcattttaccctggactcagtgtcctcctcagctttgggcaggactagatagcttgggaagaagatcctgaggaaactgtttatccttcactacccccagagcaagtataagccaaagcttgttatgaaatggcccacgatggccaaggaaaaggctcctgtgcacaacaaacgtagtctgatccttgtgtgctcgcttcttacagcatcaagaaccaaccacaggatgtatgcatgggaatcatgacaggtgagtcctgggtttccagccagactccaaatcgtccccaccccctgggacacagcagatgttttgttaataaagaagatgcactttattgcttcattgcatcactagtgactgggaagacgggaaggcatgctacggagacataagGGAGGCAGGGGATAGAAGACGTGTGACCACACGTCCTGAATAGAAAGCATGAGAAACATTCAGGGTAAGGGGGAACTGGAAGGACCAGACCAAAGCCTGTAAGATCTTGCTCCTGTATGACTACCTCAGAGGTGGATTCATGGCACTTACCTGAGAAAAATG

 

Mutation details

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47032 ACG AGG CAG GGA AGC TAC G Wild type Reverse A Wt R
47034 Fluorophore-1 AGC AAG GTG GAC TTG CTC TTC Quencher-1 Wild type Reverse
49020 CAG AGG GTG GGG AAG CAG Common A Common
49021 ACC CTG AAT GTT TCT CAT GCT Mutant Reverse A Mut R
49022 Fluorophore-2 AGA AGA CGT GTG ACC ACA CGT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47032 0.40 uM
49020 0.40 uM
49021 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.