Protocol 35737: Probe Assay - Gt(ROSA)26Sor<tm1.1(rtTA,EGFP)Nagy> Probe
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=  85 bp

Wild Type = 147 bp

>chr6:113075919-113076065 147bp CTTCCCTCGTGATCTGCAAC AATCTGTGGGAAGTCTTGTCC

Sequence

Wt Sequence: ctctcccaaagtcgctctgagttgttatcagtaagggagctgcagtggagtaggcggggagaaggccgcacccttctccggaggggggaggggagtgttgcaatacctttctgggagttctctgctgcctcctggcttctgaggaccgccctgggcctgggagaatcccttccccctcttccctcgtgatctgcaactccagtctTTCTagaagatgggcgggagtcttctgggcaggcttaaaggctaacctggtgtgtgggcgttgtcctgcaggggaattgaacaggtgtaaaattggagggacaagacttcccacagattttcggttttgtcgggaagttttttaataggggcaaataaggaaaatgggaggataggtagtcatctggggtttta

 

Mutant Sequence: GGCCTGGGAGAATCCCTTCCCCCTCTTCCCTCGTGATCTGCAACTCCAGTCTTTCTAGccttaattaagggatctgtagggcgcagtagtccagggtttccttgatgatgtcatacttatcctgtcccttttttttccacagctcgcggttgaggacaaactcttcgcggtctttccagtggggatcgacggtatcgtagagtcgaggccgctctagaactagtggatccggaacccttaatataacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagctagcttatcgataccgtcgagatcttgggtacccgctcgaaattcaccatgccaaagagacccagacccTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
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
36158 CTT CCC TCG TGA TCT GCA AC Wild type Forward A
46899 GGT ACC CGC TCG AAA TTC A Mutant Forward A vector
46900 GCT CTA ATG CGC TGT TAA TCA CT Mutant Reverse A rTta
46901 Fluorophore-2 CAT GCC AAA GAG ACC CAG ACC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
21309 0.40 uM
36158 0.40 uM
46899 0.40 uM
46900 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

Stock Number Strain Name
024746 STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy Hprt1tm1(tetO-Dkk1)Spdl Tg(TCF/Lef1-lacZ)34Efu/J
031011 STOCK Gt(ROSA)26Sortm1.1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J
2 strains use this protocol