Protocol 37251: Probe Assay - Glipr2<em1(IMPC)J>Alternate 2
Version 3.0

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

>chr4:43968866+43968968 103bp GGGAGCCTGAAGAGAGATGA CCGGCTGAAGTAATACAGATGC

 

Mut = 95 bp

Wt = 103 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 tgttgttcgttcctctgtcattccagcttccaaacagtttaataacgaggtcctgaaggCCCACAACGAGTACCGGGCCCAGCACGGTGTCCCGCCCCTGAAGCTTTGTAAGAAGCTCAACCGGGAAGCCCAGCAGTGAGTTCCCAGCAGCCATTGGCTCGGGCCCTTTGTGGAGCTGCTTCTGcagtcatgagggcccctgggtggttgaagggatgtgatggggtctcagatatagccagacttaaggacctactcttttgctggcccaggcaggcccctttgctcaatcttctaaaaatgggatggttgggaggcccaggctatacaagacctgtgtttctgttatccctctggtgaacccagatagggaaggaggctcagcagtacaaatctcagacgttggatgttggatggtgtgtgtgtcagggatggaggtgggggtgtccccgggcggggcctgctctgaggagatacattatgcaggtattcggaggccctggccagcacgagaattctcaagcacagcccagagtccagtcgtggccagtgtggagagaaccttgcgtgggcgtcctatgatcagacaggtaggacactcagctcctccccgctctgctctgctagatctaagatgaggcctctgggcgctcctcctggcactgcccttcacagttcaatgatggtagagccacaggcccaggaagggaaagaaaacccctgaattctctgcatctgttcctgggctgacctcagtagctgccccacagccagtaggctgggagcctgaagagagatgagtcagaggagacagctcgggtgcagaagtacaagtcagaggcgcgggCGCTGGAGACTGCAGCATCTGTATTACTTCAGCCGGCTGCGGGCACCGGAGCGTCCCGTGCTTTGCTAAATTATTTATTT

This mutation is a 659 bp deletion beginning at Chromosome 4 position 43,968,272 bp and ending after 43,968,930 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49988 AAG CCC AGC AGT GAG TTC C Mutant Forward A
49990 CCG GCT GAA GTA ATA CAG ATG C Common A
50770 Fluorophore-1 AGC AGC CAT TGG CTC GG Quencher-1 MUT Probe
50888 GGG AGC CTG AAG AGA GAT GA Wild type Forward A
50889 Fluorophore-2 AGG AGA CAG CTC GGG TGC A Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49988 0.40 uM
49990 0.40 uM
50888 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.