Protocol 35673: Probe Assay - Ergic1<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

>chr17:26629464+26629557 94bp GTGTTCATGGAGGGCAAATG CCACTGTGAGGGACAAAGAGA

Mutant=  109 bp

Wild Type = 94 bp

Sequence

Wt Sequence (deletions in lower case):

 GAACTGTTTGGGGTACTGTGACTCATAGCCAAGCAACCCCCTTCTCCCCTACTGGTTGACTTCCCATGTAGCCTGGGTTTCCTGGGCATCGTGTTCATGGAGGGCAAATGGGGCCTCTGTagaagcgggctgctccccactctacatgggtgtgggtaactgctctctttgtccctcacagtggtcgggcttgacatccaagatgagatgggcagacacgaagtgggccacatcgacaattccatgaagatcccgctgaataatggagccggctgccgcttcgagggccagttcagcatcaacaaggtacggagggaggcactgcctgggcccttttcctgccccctgcccactcgaccctaacagagtcagcacatgactgccgagaattttcctgagactgggtaggctctgcattccctgagattctagagcctctgatggtgggttcagaaagagctgcccctcgctggagtggaaattacCAAGGGAGGTGAGACCTGGCTTCAAGTCCTCTTCCTCCTCAGCAAGCAGTGGCCACAGGACATTGGGGTTTACAGCTGCGATGACAGCAATGCCACAGTGGCATCCACCACGGCACAAAGCCCACGGGTGCAAATGAGAGAGCACACTGTTCATTCCAGGCCTGTATACGCACCTTGCATCAGAGTCAGATC

This is a 365 bp deletion beginning at Chromosome 17 position 26,629,494 bp and ending after 26,629,858 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46707 GTG TTC ATG GAG GGC AAA TG Common A
46708 CCA CTG TGA GGG ACA AAG AGA Wild type Reverse A
46709 GCA GCT GTA AAC CCC AAT GT Mutant Reverse A
46712 Fluorophore-1 CTG CTC CCC ACT CTA CAT GG Quencher-1 WT Probe
46713 Fluorophore-2 CCT CTG TCA AGG GAG GTG AGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46707 0.40 uM
46708 0.40 uM
46709 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.