Protocol 32228: Probe Assay - Gt(ROSA)26Sor<#m1(Stop-Cas9)> Probe
Version 1.0

Notes

Mutant=  129 bp

Wild Type = 193 bp

>chr6:113025913-113026105 193bp CTGGCTTCTGAGGACCG AATCTGTGGGAAGTCTTGTCC

 

Mutant=  129 bp

Wild Type = 193 bp

>chr6:113025913-113026105 193bp CTGGCTTCTGAGGACCG AATCTGTGGGAAGTCTTGTCC

 

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

Sequence

Wt Sequence:atacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctgggagaatcccttccccctcttccctcgtgatctgcaactccagtctTTCTagaagatgggcgggagtcttctgggcaggcttaaaggctaacctggtgtgtgggcgttgtcctgcaggggaattgaacaggtgtaaaattggagggacaagacttcccacagattttcggttttgtcgggaagttttttaatag

 

Mutant Sequence: ATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCCCATCAAGCTGATCCGGAACCCTTAATATaacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagctagatcgaattcggccggccttcgaacacgtgccaccATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTAT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21306 CTG GCT TCT GAG GAC CG Wild type Forward A
21309 AAT CTG TGG GAA GTC TTG TCC Wild type Reverse A
21310 Fluorophore-1 TAA CCT GGT GTG TGG GCG TTG T Quencher-1 WT Probe
37294 TCC GGA ACC CTT AAT ATA ACT TCG Mutant Forward A
38645 Fluorophore-2 TGG TCC TTA TAG TCC ATG GTG Quencher-2 Mutant Reverse A Cas9
38646 Fluorophore-3 AGG TCC CTC GAC CTG CAG C Quencher-3 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
21306 0.40 uM
21309 0.40 uM
37294 0.40 uM
38645 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 4.0 -- 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.