Protocol 37493: Probe Assay - Gprasp2<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

>chrX:135843474+135843576 103bp GGAAGAGCAGGACTCTGTGG AAGATGCTCGCGAATTTCC

Mut = 108 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TGGGAAGGTACAATACTGTGACAGACCTGTCATTGAGACTGAGGCTTTGGTAGGCATTCTTAAATTGAGCCTTCTAACTTGTACCATGACTGGGGCAGAAGTTGagaccggttctcaggccaagcctgataagaagcctcaggaagaggtcgcaggtggggctgagagagaaagtgaagcccctctggtggtcagacccaaagtcaggcctcaggctccagcaacaagtggtgctaggcccaaaactgaaactaaatcttcctctcgggcaaggcctaaaactgaaacccagtcagtgtctggaacaagacaccgaatgagtggagcaaggccgagatctgaggctcaattaatgtctggggccaggcctaaaaccgatgccagggcagtaggtggtgctcgccctaagactgaggccaagccaatcccaggagcaaggcctaagggtgatgcccaggcttgggcccacagtgagtttggagccgaggcaatgccacgtgctgagagagcacacttatctaattctgtcacatggccaccagtcaatgttgggtctgcaactgttactaaatctaagagtctgtctatgaatacagaactggccagtatgggtagtgaaatcttttctggcacccagggtcagcctggaatcgagccctggtttggaccgagagaagaggctaatatgggttcttggtgctatcccaggcccagagccagagaggagacctctaatgagtctgcagatgaaaactctacaatgtcttctttctggactagagaggagactagtatcagatcatggcccagggaagaggtgaacactaggtccaggcacagggctaaacatcagactaatgccaggtcgaagcccagatccaaacaagatccctatattgattctttgtctgggtccgaggatgaggccagtaacccattctgcttttgggctggagaaaatactaatgacatgtttagggccagaggcagggatgaggcgaatgccagacccaagatcaggacaaagagagaggactattttgaagatgaagatgagatctataaagagtcctggcttttgcctggggaagagggtaatagatttaggcgccgagacaaggaagagcctaataagaccttgaaaaatgagaacgaaaaagatgttaaaaatgatgaaacagtcgaacaagagtccaggcttgaggaggaagtcattattgggtcctggttctgggcagaacaggagactaatgtggaggctggagcttcagcaatctgtgacgctgaaccaggggctgaagagggggccattggtggatccctgttctggaccgaagaaaagcctagtttgggggctgtggccagagatgaggtcaggcctgagtctgaagaagaggcactatttgggtcctggttttgggatagggatgaggcctgctttgacccaaatcctactcctgtgtacacagctaagagcaggtacagagatccagaagaggatcttaatttagcatccaggcccaaaacctgggacgaggttaccatcgaattcaaacctccttgccatgggcttgggttcccttccccaagaccctttatcattcctgaaggggcttctggaaatagtgaggaaaaagccaagaatgcagagcttggcgcagagggggaagagcaggactctgtggctcagcgtgatcttcctgaacccgagtttccatttcagtatgatccctcttaccgctcagtccaggaaattcgcgagcatcttaaggccagggaaagtgcacagccagagaattggtcttgcacctgcatccagtgtgagcttagaattagttctgcagaatttgaggagcttcttttactgatggacaggattcgtgatccttttatccatgagatagctaagattgcaatgggcatgagaactgcttctcagtttacccgggatttcattcgcgattcgggtgttgtctcactgattgaagccttgatgaattatccttcctcccgagtgaggactaattttttggaaaacatggttcacatggctcccccttatccaaatctaaacatgattgagacatttatttgtcaagtgtgtgaggagacactttcacatagcgtgaattcccctgaacagctaactggaatgaggatgcttagacatctcactataactactgactatcacgtactgattgccaattacgtgtctgggtttctcgccttattaaccacaggcgatgcaagaaccaagtttcatgttctgaaaatgctgttgaacttgtctgacaatccgatggtggcaaaaaaactattcagtgccaaagctctatcaatatttgtgggtctctttaacatagaggagactaatgataacattcagattgttattaaaatgtttcagaacatcagtaatattgtaaaaagtggagcgatgtctttacttgatgatgatttcagtcttgagccgcttgtttctgcatttcatgaatttgaagagctagctaaacagctgcagatccaaatagacaatcaaaatgatcccgaGGAGGGACAGTAAAGTTAGTGTGATTTAACCACCTGCCACTCATCAGCCTTATGTTCCCAAAGAGTCCCAAATAGTGCTTTGGTGTTC

This mutation is a 2449 bp deletion beginning at Chromosome X position 135,841,913 bp and ending after 135,844,361 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51466 GGA AGA GCA GGA CTC TGT GG Wild type Forward A
51467 AAG ATG CTC GCG AAT TTC C Wild type Reverse A
51468 TGA GGC TTT GGT AGG CAT TC Mutant Forward A
51469 TGA GTG GCA GGT GGT TAA ATC Mutant Reverse A
51470 Fluorophore-1 CTT CCT GAA CCC GAG TTT CC Quencher-1 WT Probe
51471 Fluorophore-2 ACT GGG GCA GAA GTT GGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51466 0.40 uM
51467 0.40 uM
51468 0.40 uM
51469 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.