Stock No: 034159
Protocol 39588: Probe Assay - Rag1<em10(indel)Lutzy>-alternate1
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=  107 bp

Wild Type = 82 bp

>chr2:101643577-101643658 82bp AAAGAGACTTTGGTGCATATCAA GATGTTTCTGCGCCCTTCTC

The Mut probe (primer 54171) anneals over the nucleotide sequence containing mouse genomic variations rs27362105.

Sequence

Wt Sequence (with deletion in brackets):

GTGGAAAGGGAATTAAATTTCACATATACATGTATTTAAAATCATGCATATATTTAGTATAAGTGTCCCCAAAT[attgtcaggaaaggattggccattttggctgtatgatcagcacccaacagagaatattaaaataggagatttaataaatattgttggaaagaattaaatgaatgaaaaactgagggcatcaatcagttatttccaaaatattgcgatacgctattgatacttttcttttttgtttttgtttgcttgtttgattcttttcttaacttgactcccccccccccccccattgttcccaggtagcttagccaacatggctgcctccttgccgtctaccctgagcttcagttctgcacccgatgaaattcaac……..cggggcctgggcaagtgagggaaatgaatcgggtaacaagctgtttagacggtttcggaaaatgaatgccaggcagtccaagtgctatgagatggaagatgtcctgaaacatcactggctgtatacttcaaaatacctccagaagtttatgaatgctcataacgcgttaaaaagctctgggtttaccatgaactcaaaggagaccttaggggaccctttgggcattgaggactctctggaaagccaagattcaatggagttttaaataggatctccacatagaagttggtatttgccaatgtgttttcctttgggttgcagtgaggtcttctcctagcacctagcacattgccatgtgggtgggtcttatcacccaaggggtgacatgttggagtgagaggccagaggtgctgtcacct]GGTCAGGGATCGATAGTTGACAAGATGAGATTGCCTGAGCCGTTTAGTGAGGCCAGAAGAGCAACAGGAGAAATCAGTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53511 AAA GAG ACT TTG GTG CAT ATC AA Wild type Forward A
53512 GAT GTT TCT GCG CCC TTC TC Wild type Reverse A
53513 Fluorophore-1 CTC GCC AGC ATC TCC TGT C Quencher-1 WT Probe
53514 AAA GGG AAT TAA ATT TCA CAT ATA CA Mutant Forward A
53515 CAG GCA ATC TCA TCT TGT CA Mutant Reverse A
54171 Fluorophore-2 CCC AAA TGG TCA GGG ATC GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53511 0.40 uM
53512 0.40 uM
53514 0.40 uM
53515 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.