Protocol 31034: Probe Assay - Pten<tm1Hwu> Probe 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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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=  139 bp

Wild Type = 173 bp

Sequence

Wt Sequence: cccagagctgagcatcctaagcaagcactctgcgaactgagctacatcccagagttcataccaggatttaaggatctcaataggatagaatcaaaacagatactagtaagataaaaaccagtagtgatagaacggaagtcttgcttctaGAtaatagcatcttgccttcaaaaacttaactctgactatagagaacaaagacatcttagattcttaattcatgtg

 

Mutant Sequence:aaacagatactagtaagataaaaaccagtagtgatagaacggaagtcttgcttctagTGCGGCCGCACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCCGATCATATTCAATAACCCTTAATataacttcgtataatgtatgctatacgaagttatTAGGTCCCTCGAAGAGGTTCACTAGataatagcatcttgccttcaaaaacttaactctgactatagagaaca

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35320 GTT AAG TTT TTG AAG GCA AGA TGC Common A
35329 Fluorophore-1 CGG AAG TCT TGC TTC TAG ATA ATA GC Quencher-1 WT Probe
36015 CTG AGC ATC CTA AGC AAG CA Wild type Forward A
36016 CGA GGC CCT TTC GTC TTC Mutant Forward A
36017 Fluorophore-2 AGG TCC CTC GAA GAG GTT CAC TAG ATA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35320 0.40 uM
36015 0.40 uM
36016 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

Stock Number Strain Name
033964 B6.129(Cg)-Ptf1atm2(cre/ESR1)Cvw Krastm4Tyj Ptentm1Hwu/J
033754 STOCK Brca2tm1Brn Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ
033752 STOCK Gt(ROSA)26Sortm1(TMPRSS2/ERG)Key Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ
033763 STOCK Gt(ROSA)26Sortm2(myc*T58A)Rcse Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ
033753 STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Brca1tm2Cxd Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ
033757 STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu Tg(ARR2/Pbsn-MYC)7Key/AbshnJ
033751 STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ
033755 STOCK Gt(ROSA)26Sortm3(CAG-EYFP)Hze Trp53tm1Brn Nkx3-1tm4(cre/ERT2)Mms Ptentm1Hwu/AbshnJ
8 strains use this protocol