Protocol 44059: Probe Assay - Txnrd3<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

>chr6:89651669+89651787 119bp TGTCAAAGGGGCATAGCAGT CCTGGCAATCAGCATTGTA

Mut= 101 bp

Wt= 119 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TTAAAGGACACTTGTCCTTGGTCTCAGCACAGCTGTTGAGTATATAACCCAGATGCTTTGTTTTGAAAGGAATCCATGGGTGGGCCTTTGGGTTTCAGGTGTCCTCTGAAGGCATCACTGTTTTCCCATCTTAAGTCATTATTTCATAAACGCATGGAGCACTGGGCTTCATGGATGTGAACAGGCAAGCCATGAGCAATGGAGCACTGGAAGGCTTATGCTCTgccaggggccctttgtggtatcttcatatattgttttattccatagatgacggggccagtgttcaggaagtgctgacagaaatcagtaaccagaaaacggtgcccaatatttttgtgaataaagtgcacgtgggtggatgtgaccgaactttccaggtaatgatggtcatcagacacggtcctcaacactgtgtgttcacattgcaatttaatccatgtcaaaggggcatagcagtggaaaggcacaggggctgggtatgggataatcctgaagatgcaagggaaatgagctTTTCCATCAATACCTGAGAAGTTATACAATGCTGATTGCCAGGCACAGGAGGGACATTCTTGGCCTGAAGTCAGAGCAGCTGTGGCCCTCATAGAACTGAGTCTACTACCACTCCTCTATGTATGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGAGTGATCATGAGTCCTGTGAGGGATTCATGGTGGCTCAGGAGCAGCTCATGACTCATGGGG

 

This mutation is a 292 bp deletion beginning at Chromosome 6 position 89,651,453 bp and ending after 89,651,744 bp (GRCm38/mm10). In addition, there is a 23 bp TTACCAGGCCCTGCCCAAATTTC insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64680 TGT CAA AGG GGC ATA GCA GT Wild type Forward A
64681 CCT GGC AAT CAG CAT TGT A Common A
64682 CCA TGA GCA ATG GAG CAC T Mutant Forward A
64683 Fluorophore-1 CTG AAG ATG CAA GGG AAA TGA Quencher-1 WT Probe
64684 Fluorophore-2 AAG GCT TAT GCT CTT TAC CAG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64680 0.40 uM
64681 0.40 uM
64682 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.