Protocol 34123: Probe Assay - Mrgprg<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

>chr7:143764495-143764605 111bp CAGCTCAGGAAGGACCCACT GGCCACTCTCTACACTTGGTG

Mut= 116 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GACTCTGGAAGCTGCAGGCTCTTGCTTTCCACACTCGGGGCACAGCAGCTTCGTAGTGAACACTGTCACCCTTTGCAGGGGCCTCCCAGAGCACCCACGGAAGCCAGCCAGGATGTTCTCCATattcaacatctggggcacattcaacaaagtgctcttcttcctcagcctcactgtcagccttgcagggctggtgggcaacgctctgctgctctggcacctcggtctgcacattaagaagggacccttcaacacctacctgctgcacctggctgctgccgatttcctcttcctctcatgccaagttggcttctctattgctacgatcgtgtcgggccatgaggacacactctacttcccagtcaccttcctgtggtttgccgtggggctctggctgctggctgccttcagtgtggactgttgcctcgcctacatgttcccctccttttgcagccccaaccgccgtcccaggttcacttcagttgtcctctgcctcgtgatctgggccctgaccatgccggccgtgctgttgccagccaacgcctgtgggctgctaaaaaacggaatgagtctgctggtctgcctcaagtaccattggaccagtgtcacctggctagcggtactgtctggcatggcatgtggggcaagcaagtttctcttaatctttgggaactgctgctcttcacagccaccccccaagttctgcaagttggcccagtgctctgggatccttctgttcttctgtcgcctacccctggttgtctactggtgcctgcggccagtcttaaaattcctgctccccttcttctttccactggccaccctcctggcctgcattgacagcagtgcaaagcccctcttgtactacatgaagggcaggcagctcaggaaggacccactgcaggtagccctaaacagggctctaggggaagagtcccagtcAGGTCTTGGGGGGCTATCCTTGCCCATGCACCAAGTGTAGAGAGTGGCCATTATACGCCCACCTACCCTGCCCTCAGAGCTGCCAGGGGGTCCCAGTCTTGCTCAAACCTTTGCG

This mutation is a 819 bp deletion beginning at Chromosome 7 position 143,764,544 bp and ending after 143,765,362 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42883 CAG CTC AGG AAG GAC CCA CT Wild type Forward A
42884 GGC CAC TCT CTA CAC TTG GTG Common A
42885 TGA ACA CTG TCA CCC TTT GC Mutant Forward A
42886 Fluorophore-1 CAG GGC TCT AGG GGA AGA GT Quencher-1 WT Probe
42887 Fluorophore-2 CCA GGA TGT TCT CCA TAG GTC TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42883 0.40 uM
42884 0.40 uM
42885 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.