Protocol 46850: Probe Assay - Tpbgl<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

Mutant=  121 bp

Wild Type = 141 bp

>chr7:99626189-99626329 141bp ACATCGAGGTAGTAGAGGAC CGTGATTGAGCTGTAGTGAG

Sequence

Wt Sequence (deletion in lower case):

AGCTGGAGGACGGACCGGTCGGAACTGTCAGCTGCTCCGGGTCAAGGTCTAGCCCACAGCCACCTTCCCGTGCCACCCCCATCCCATCATCTCCAAACGCTCCCAATTCACTGGTGAGCGCTGCGGGCCGGGGGCTGGATGCGGGGACGGCCGCGATGGCCCCGCGCGCGGgacagcgggggctctggagcccgctgccagggctgctgctcttggcggcggcgctgagccggcccgccgcgccctgtcccttccagtgttactgcttcggcagcccccggttaatgttgcgctgcgcgtcgggcgcggagctccggcagccgccccgggacgtgccacccgacgcgcgcaacctcaccatcgtgggcgccaacctgaccgtgctgcgcgccgcagccttcgcgggagggggcgagggggcgacggacggcgtgcgcctaccgctccttaccgcgctgcgcctcacacacaacaacatcgaggtagtagaggacggtgccttcgacgggttgcccagcctggcggcactcgacctaagccacaacccgctacgcgccctgggctaccgcgcctttcgcgggctgcctgcgctgcgctcactacagctcaatcacgcgctggctcgaggcagccccgggatgctggatgcactggacgccgctctggcccccctggccgagcttcgcctgctgggcttggtaggcaacgcgctgagccgcctgccactcgccgcgctgcgcctgccccgcctggagcagctggatgcgcgtgtcaacgcgctggccggcctgggcccggacgagctgagcgcgcttgagcgcgatggcgacctgccccagccgcgcctgctgctggcggacaacccactgagctgtgggtgcacctcgcgccccctgctggcctggctgcacaacgccaccgagcgcgtacccgacgcgcgccgcctgcgctgcgcttccccgcgcgtactgttggaccggcctctgatagacctggacgaggcgcgactaggttgctccgacggcgatgcacacgagagcggggaagggatagacgtcgccggcccggagttggaagcctcttacgtcttcttcgggctggtgctggcactcatcggcctcatcttcctcatggtgctctacctaaaccgccgcggcatccaacgctggatgcacaatttgcgcgaggcctgcagagatcagatggagggctaccactaccgctacgagcaggatgcagacccacgccgcgcgcctgctccggccgcccctgccggctcccgggccacttctccaggctcggGTCTCTGAGCATCACCTCCCTAGTGGGAGGCTGTTCCTATCAGCTGATGACTTTACTTGGGCCATGCAGCCTTTCTCTGCCTGGCCTGGGCCCCGGAGATAACACACTTACGAGTTTGGATCTCTGAGACCTTTGGATCAATCGTAGTATCTCTCCCATACAGAAATTCCAAGGTGTACTCTTTCCCTGGCCTTAGAGTTTCCAGATCAAAAACCCAGAAATCTGTTTTCAGAAACAAATCCTGCCTGCCGGTCCTTA

 

This mutation is a 1131 bp deletion beginning at Chromosome 7 position 99,625,502 bp and ending after 99,626,632 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
69011 ACA TCG AGG TAG TAG AGG AC Wild type Forward A
69012 CGT GAT TGA GCT GTA GTG AG Wild type Reverse A
69013 CCC AAT TCA CTG GTG AGC Mutant Forward A
69014 GTC ATC AGC TGA TAG GAA CA Mutant Reverse A
69015 Fluorophore-1 ACT CGA CCT AAG CCA CAA Quencher-1 WT Probe
69016 Fluorophore-2 CGG GTC TCT GAG CAT CAC CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
69011 0.40 uM
69012 0.40 uM
69013 0.40 uM
69014 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.