Protocol 44165: Probe Assay - Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas-Chr3-Probe Alternate2
Version 3.0

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=  151 bp

Wild Type = 154 bp

>chr3:62978528+62978681 154bp ACCCCCATGTCAGAGTTCCT TGAGAGACTCTTAGAACTCAATAGG

Sequence

Wt Sequence: acccccatgtcagagttcctgactgtgggatggcaaccccatccctccacttgatgtcctgtgtttctactggaggtggacttcacaaattccctccctctactgtTGGGCATctcatccaaagtccctcctattgagttctaagagtctctca

 
Mutant Sequence:
ACCCCCATGTCAGAGTTCCTGACTGTGGGATGGCAACCCCATCCCTCCACTTGATGTCCTGTGTTTCTACTGGAGGTGGACTTCACAAATTCCCTCTCTCTACTGTTggcGTAATAGCGAAGAGGCCCGCACCGATCATAAAACGGGTTTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37598 ACC CCC ATG TCA GAG TTC CT Common A mChr3
37815 CAA ACC CGT TTT ATG ATC G Mutant Reverse A vector
37817 TGA GAG ACT CTT AGA ACT CAA TAG G Wild type Reverse A mChr3
37818 Fluorophore-1 TTG GGC ATC TCA TCC AAA GTC Quencher-1 WT Probe
64908 Fluorophore-2 TGT TGG CGT AAT AGC GAA GAG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37598 0.40 uM
37815 0.40 uM
37817 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
033265 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD100/RwwJ)F1/J
033245 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD2/TyJ)F1/J
032882 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD32/TyJ)F1/J
033248 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD42/TyJ)F1/J
033254 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD61/RwwJ)F1/J
033255 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD62/RwwJ)F1/J
033256 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD65/RwwJ)F1/J
033257 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD68/RwwJ)F1/J
033258 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD69/RwwJ)F1/J
033259 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD70/RwwJ)F1/J
033260 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD75/RwwJ)F1/J
033261 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD77/RwwJ)F1/J
033262 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD81/RwwJ)F1/J
033263 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x BXD87/RwwJ)F1/J
032883 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x C57BL/6J)F1/J
032881 (B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax x DBA/2J)F1/J
16 strains use this protocol