Protocol 37956: Probe Assay - Eid2b<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

>chr7:28277753+28277831 79bp CGGTGCTTCTGAGGATTCA GTTCATGGGGCTCAGGTCC

Mut= 80 bp

Wt= 79 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AATAATAATAATAATAAATTAAAGTATGTGATGCCGCCCCTGCTGGTTCAGAACTTGAGGCGGCCGTGGCGGTAATGGCACATGGCACCCCTAGAGGGCGATAGAGGTCCGGGCACCCGTCCTTGCGCAAGCACTTTTGTTTTCCAGGGCGCCGAGGCCCGCGCATGCGCACATCTTCACGCTGCCGCCGGAGCCGGTGCTTCTGAGGATTCAACGGTGGCAATGTCCGAGCTTCCcggagagagcaggatcccggacctgagccccatgaacggcgtcggcgacgtcctgcaggtgggactgggcggaggcagtccggtccccgaagcccaggaagacccgctggccacagcctcccagcccatggcgccggccccgggcctggctcccgggcagttccccggcgtggcgggcctgatggcgatcccgcacgtccacgcgccgctcctcctcctagagctgtacgagcagcggatgttccaacactatctgcacaccaaccccctgatccccaccaggctgctccacgaaatcgaggagcgccgccggctgttcgtggagggctgcagggccagggaggcggccttcgacgccaaccccccgcagatggactcgcacgcccgcgccttcatgctggcgctcacggcctccgacgcccgcggccccaaCGCCGACTGAGGGGTGGCCTTCACCTATAACCTCCGAGGAAGGAGGCCGGAGGACCGGACTTCAAGGTCATCCTCCGCCATGTAGCGAGTTGGAGGCCAGCCTGGGCTACAGGAGACTGTCTTTTAAAAATAAAAAATAAAAATAAAAGATTGGTGGCCAGGAAGTCAGGTGACTCCTAGAATCAAGGGCAAAGGCAGATGTCTGGGCAAATGTCATGAGGTGGGGAATCCATGCCAGAATCCA

This mutation is a 432 bp deletion beginning at Chromosome 7 position 28,277,795 bp and ending after 28,278,226 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52615 CGG TGC TTC TGA GGA TTC A Common A
52616 GTT CAT GGG GCT CAG GTC C Wild type Reverse A
52617 CTC GGA GGT TAT AGG TGA AGG Mutant Reverse A
52618 Fluorophore-1 ACG GTG GCA ATG TCC GAG Quencher-1 WT Probe
52619 Fluorophore-2 AGC TTC CCG CCG ACT GAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52615 0.40 uM
52616 0.40 uM
52617 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.