Protocol 36522: Probe Assay - Tubal3<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

>chr13:3933373+3933515 143bp GTTGAGCAACACCACAGCA GATCTTCCCTGGCCTCTACA

Mut= 140 bp

Wt= 143 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCCTCCCTCCCTCCTCTCTCTTTCTGAAGATCAAACTCAGACCTGCATGCTTGTAAGGCAAGCACTTTACTGATGGAGCCTTTGCCCTAGCCTCCTTTCTGAGTCCTTTGTTCTTGGGAAGCTATGTTCTACCATTATCATGGTAAACAAACAAGAAGGCACTTTCAGAGTTTGGATATGAGTATTCTTCAAGTAAGAAAGTACTGATTCCTTCTAGTTAACTGACTGTAATTCTCACTCCCCCCCCCCCGCCCCCAGgcagaacaatgcagtggacttcagggatttttgatttaccgaagctttggaggaggcacagggtcgggatttacatctctcttgatggagcgactctccgtagagtattgcaagaagataaaattggagttctctgtctacccatctcctaggatctccactgccgtggtagaaccgtacaatgccatcctcaccactcactccaccatagaatactcagattgtgctttcatggtggacaatgaggctctgtacgacatctgccagcacaaacttggcattgaacgtccttcctatgctagtatcaacaggctgattgctcaggtgtcgtcttccatcactgcatcactgcgttttgaaggacctttgaatgtagacttaattgaattccagaccaacctagtaccttatccaagaatccatttccccatcacagctcttgcccccatcatctccgctgaaaaggcgtaccaagagcagctctctgtgtctgacgtcactgcttcttgctttgaggtctccaaccagttggtcaagtgtgatcccaggcttgggaagtacatggcttgctgcttactgtacagaggagatgtggtccctaaggatgtaaatgaagcaattgcagccatgaaatctaggacctctgttcagtttgttgattggtgtccaactggtttcaaggtgggcatcaattatcagccacctgcagtggtaccaggaggggatctagctagggttcagcgtgctgtgtgcatgttgagcaacaccacagcaattgtggaagcctgggcccgcctggaccacaaatttgacctcatgtatgccaagaaggcattcctgcactggtacatcacagaagGCATGGAGCTTGGGGAGTTTGTAGAGGCCAGGGAAGATCTGGCTGCTCTGGAAAAAGATTATGAAGAAGTGGGTCTGAGTTTCTGACTCTTTGTAAGTCTGGTTTTTCCTTTATTCATGTGTCTATGTGAGGTGTGTGTGCTTATTTGCATATGTGCACTCATATGTGGGGAAGCACGTGTATGCACAAATGTATGTGCCCATGTGTGCACAAGTGTGTGGAGGGCAGATGCTGAACTGGGTGTGTGCCTTCCTT

This mutation is a 859 bp deletion beginning at Chromosome 13 position 3,932,618 bp and ending after 3,933,476 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49005 GTT GAG CAA CAC CAC AGC A Wild type Forward A
49006 GAT CTT CCC TGG CCT CTA CA Common A
49007 GGC ACT TTC AGA GTT TGG ATA TG Mutant Forward A
49008 Fluorophore-1 CCT GGA CCA CAA ATT TGA CCT Quencher-1 WT Probe
49009 Fluorophore-2 CTG ACT GTA ATT CTC ACT CCC CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49005 0.40 uM
49006 0.40 uM
49007 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.