Protocol 32160: Probe Assay - Slc36a2<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

>chr11:55181709-55181797 89bp GAGACCCAGATGTGCCAAAG CGTGCCAGCACTTCAATTAC

Mut= 92 bp

Wt= 89 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence: (deleted region in lower case)

 

ATAGACAAATTATCTAAGTATTTTGGGGCCTCCATTTCCCATCTACAGACCAAACCCCAACCATTGCCTACTTTATTGTCCTGCCAAGACGATTAAATGAGATGTTATATTTAAAGACTCCACGCATACAGGGTCTCATACAATTAGTGTTTCATGGTACAGACAGGGCCCAAGCCGCTAATGGAACAATTCTGGATATCTTGGGATGGGTGGTCACCTTCCAGGAGACCCAGATGTGCCAAAGCAAGGATACAGGAAAAtcgagatggccatgaatttacctgcccacgcaggtaattgaagtgctggcacgctatgaggggatggctgctaggtcctttcccagctccatttccatgtcctctgactgccttcctttcctgtagcgggttccagacattggttcatctggtcaaaggcaacatgggcacagggatcctcggacttcccctggctgtgaagaatgcgggcatcttggtaagaggcatggctaagtgggggcactacgaagaagctgtgggatagggaagaagacataatggtttaaaataccttgtagcagtcagtgcttggggggtggagaggtatctgacccagacataaaatctgaacatcaagtctgagtctgagttctgccttggcttggcctctggcattaggagtcaagtcaacactttttatccaccctcaggcttcaaatccctccttttgtagaatgtaaggattagaggaggaccagTTACCCAGTCTACCTTTACAGATGCCCTTGGGGACATCTCAGAGACCACCAATGGGCCATGTGATCAGGACCCTGAGTCTCCTGCTCCATCACAAATGTCACCGTGCTTGCTATGTGTACTTTTTAGACATGACTTGAGGTTAACTTTCGGAAAGAGTAGCCAAGTCTCTTTCAACTTGGTTTTCTAGAAAGTTCTTTCAGGACTAGCAAATGGCTCACGAGATAAAGGCACTGGCCACCAAGTC

This mutation is a 479 bp deletion beginning at Chromosome 11 negative strand position 55,181,761 bp and ending after 55,181,283 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38476 GAG ACC CAG ATG TGC CAA AG Common A
38477 CGT GCC AGC ACT TCA ATT AC Wild type Reverse A
38478 CCC ATT GGT GGT CTC TGA Mutant Reverse A
38479 Fluorophore-1 AAT TTA CCT GCC CAC GCA Quencher-1 WT Probe
38480 Fluorophore-2 CAG ATG CCC TTG GGG AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38476 0.40 uM
38477 0.40 uM
38478 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.