Protocol 34784: Probe Assay - Tex28<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=  100 bp

Wild Type = 98 bp

>chrX:74152402-74152499 98bp GGGTTACTCACTGCCAGCAT TCACCTGTTTTTCCATCAGTG

Sequence

Wt Sequence (deletions in lower case):

TGGGTTGAGATTTAATTTGCATGTCATGAAACAATAAGCCCTACTAGCAGTCATTCCTTGTCACTCCTGGTTCCCAGTTCCTGGCACCCACTCATATGACTTGTTTGTGGATTGCATACAAATGGAACTTTACACAATGAGGTCTTTATAATAAATATTCATTTATGGAGTAGATACTGCATCTTCAACACTGCTCCTGGGAGCATTTGGGGGGATAGCTTAAAATTTAATTTCTATACTTTCTAGTCTATTGCACACTTGGTAAAATAGTAGAGAATAATTGTTAATAAGATCTATTAGTGAAAGTAGTGTAAGTATTCCCAGGCAGAGATAGAAGATCCAGCCCCCTTTGACAGAATCCTAGGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTTTGTGTAGGGATGTTGTTAGTAAATCTAGCTTCGGGTTACTCACTGCCAGCATGAGCTCTGAGTAAAggtacagagggagtgttactttctctcttgcatcctagacaatcactgatggaaaaacaggtgaatgatcacttgcagagatacctggatgagatttgccacctcaaacagcatctggcatgcactgaagagaaaatggcctatttgtcctatgaaagagccaaggaaatatgggtgagggcactggggagaaaggactagtgagaaggatatggggatgaatagatgtatttggatatgcccaagccaagacaagagaagatatagagctgtgtctggcctgacagtacaatagtaacctggtttagagccagcagtgtccaccctgcatgcctgtgtgaactccactccttaccatgcagccaaggtgctttggagactttggacagtggttctaatcttgtagggtcaattactgactcataccctggaatctgtgtcctataaatcaggagtgaggcctgacaaacccaaggaatgctggggtgtttgcattgctagtagagagaaAGCCTTTGGGAATCTCTTCTTGGGGTCTAGCTTTTTGCCCCTCAGTCTTTTCCATACTCATAGCTCCTACTCCTGTTCCTGGTTAGACCTTTTTCACCTAGGTTTTCAGTCTCAGCAGTAGATAGTTCTCACCAGGACCCATCAGCCCAGCATTCCACAGCAATAGTCTGTCTCTGCTGGGCATACCCAAGAGGTTCTTCCCATCTTTTCTAAAAAATTCAACTCATCAGGTATGATGGCACATGCCTTTAATTTCAGGACTTGGGAGGCAGAGGCAGGCAGATCTCTGAGTTTGAGAACAGCCTGGTCTATCTAACAAGTTTTGGCTCACCTGGAACT

 

This mutation is a 510 bp deletion beginning at Chromosome X position 74,151,956 bp and ending after 74,152,465 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44441 GGG TTA CTC ACT GCC AGC AT Common A
44442 TCA CCT GTT TTT CCA TCA GTG Wild type Reverse A
44443 GAG CTA TGA GTA TGG AAA AGA CTG A Mutant Reverse A
44444 Fluorophore-1 AGG TAC AGA GGG AGT GTT ACT TTC TC Quencher-1 WT Probe
44445 Fluorophore-2 AGC CTT TGG GAA TCT CTT CTT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44441 0.40 uM
44442 0.40 uM
44443 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.