Protocol 34971: Probe Assay - Human APP Indiana (V617F)
Version 1.0

Notes

This assay cannot distinguish hemi from hom

 

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

Tg=  96 bp

IPC = 74 bp

>chr16:84962983-84963078 96bp CGCCATCATCGGACTCAT TGGATGGATGTGTACTGTTTCTTC
These "transgene" primers amplify mouse.  The probe is what makes this assay specific.

Sequence

Tg Sequence:

 

 

 

Indiana (V617F) 642 of 695aa

Hu WT V        TTGTCATAGCGACAGTGATCGTCATCACCTTGGTGATGCTGAAG

Hu V->I        TTGTCATAGCGACAGTGGTCATCATCACCTTGGTGATGCTGAAG  I716V(FL),V717I(Lon)

Hu V->F        TTGTCATAGCAACCGTGATTTTCATCACCCTGGTGATGTTGAAG  Indiana allele

Mouse B6J      TTGTCATAGCAACCGTGATTGTCATCACCCTGGTGATGTTGAAG

                        A  T  V  I  V  I  T  L  V  M  L  K

 

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45231 Fluorophore-1 AAC CGT GAT TTT CAT CAC C Quencher-1 MUT Probe
45232 CGC CAT CAT CGG ACT CAT Transgene Forward A
45233 TGG ATG GAT GTG TAC TGT TTC TTC Transgene Reverse A
oIMR1544 CAC GTG GGC TCC AGC ATT Internal Positive Control Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45232 0.40 uM
45233 0.40 uM
oIMR1544 0.40 uM
oIMR3580 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.