Protocol 21980: End Point Analysis Assay - Hbb<tm2(HBG1,HBB*)Tow>/Hbb<tm3(HBG1,HBB)Tow>-EP
Version 2.0

Notes

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 = TM2/TM2
Heterozygote = TM3/TM2
Wild type = TM3/TM3

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
18872 GAA GGA GAT TCA TCC ATG CAC Common A
18873 AGC TCT GGG TAC TCC CTC TGA Wild type A
18874 TCC CCT AAG CTA TCA GGT TGA Transgene A
18875 TTG TTC CGA TTC AGT CAT TCC Transgene A
18876 Fluorophore-1 TTG TCC TTA TCT GTG CAG AGA ACA Quencher-1 IC Probe
18877 Fluorophore-2 GGT GAA ATT CTT TCA TCC TTC ATG Quencher-2 MUT Probe
18878 Fluorophore-3 CTT CCC TTT AGC TAG TTT CCT TCT C Quencher-3 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
18872 0.40 uM
18873 0.40 uM
18874 0.40 uM
18875 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
027265 B6.Cg-Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow Hbatm1(HBA)Tow/ShmtJ
013071 B6;129-Hbbtm2(HBG1,HBB*)Tow/Hbbtm3(HBG1,HBB)Tow Hbatm1(HBA)Tow/J
2 strains use this protocol