Protocol 36282: Probe Assay - Slc7a3<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

Mutant=  129 bp

Wild Type = 131 bp

Sequence

Wt Sequence (deletions in lower case):

AATGGGTCCTTCCACCTGCCTGGGAGACACTGTAATCCCCACTTGCATTATTATTGTAATTCCTACTTGCATATGAAGAAATGGTAATTAGAGGTGGAAGTGGCTTGCTTTCTCCTCTTCTGCCCAACCCCCTTTGTCCCTGTAACTCATTCACCTGGTGGAGGGAGTGGGTTTTGTAATTCACTTTTGGAGCTTGTGGGGGACAATGAGACCCATTGAAGGGCTTTTGTCTTGTTTTTCACCAGAACCTAGCTAGCATCCTCTTCAATTCCAACTAGAATGCTGTGGCAGGCACttcgaagatttggtcaaaagctggtgcggagacgtgtgttggagctgggtatgggtgagacccgccttgccagatgcctgagcaccctcgacttagtggctctgggtgtgggcagcacattaggtgcaggtgtgtatgtcctggctggagaggtggccaaagataaagcaggaccatccattgtgatctgctttttggtggctgctctgtcctctgtgttggctggactgtgttatgctgaatttggtgcccgggtccccggctctggttctgcatatctctacagttatgtcaccgtaggtgaactctgggccttcaccaccggctggaatctcattctctcctacgtcattggtgagatagatgcttggggtaggatgggagggatggatgattacacaaatagcagagggattatggatccagaaatccgagtgcaatTTCAGTGAAGTCTAAATTGTCTTTATTTTTCCTAGTATCTCAATACTTCTAGATGTCTGTTTGTTTGTTTATTTATTTATTTATTTGGGGTAGGGGTATAGGGAGGGTTTGAAGGTGCAGTACAATTGTTCACCTCCGTCTGGTGTTCATTCATTAAGATGTTAGTTAGGGTCTTTAAAATTAATAACAGGAGCTGGGTCTAGAGTTGTACACCTATAATTCCAGCACTCAGAGGCTAAGGTAGGAGGAT

 

This mutation is a 441 bp deletion beginning at Chromosome X position 101,083,784 bp and ending after 101,084,224 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48309 CTT CAA TTC CAA CTA GAA TGC TG Common A
48310 CAC TAA GTC GAG GGT GCT CAG Wild type Reverse A
48311 CCC CTA CCC CAA ATA AAT AAA T Mutant Reverse A
48312 Fluorophore-1 AGC TGG TGC GGA GAC GT Quencher-1 WT Probe
48313 Fluorophore-2 AGG CAC TTC AGT GAA GTC TAA ATT GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48309 0.40 uM
48310 0.40 uM
48311 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.