Protocol 43904: Probe Assay - Ap5s1<em1(IMPC)J>
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=  100 bp

Wild Type = 133 bp

  
>chr2:131213472+131213604 133bp CCAGGTGATGCCTTATTTTG GTCTTAGAAGGGTGCAGCAG

Sequence

Large deletion:  Mutant sequence with junction in uppercase

gcccttgttctgtgatgcagagcaggcactagattatTTgagtgggagtctgctgggtggccccgatctcctcctccctgctgctgcacccttctaagac

This mutation is a 2400 bp deletion beginning at Chromosome 2 position 131,211,143 bp and ending after 131,213,542 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64275 CCA GGT GAT GCC TTA TTT TG Wild type Forward A
64276 GTC TTA GAA GGG TGC AGC AG Common A
64277 GCC CTT GTT CTG TGA TGC AG Mutant Forward A
64278 Fluorophore-1 TGG CGG TCT GAT CTT GTC C Quencher-1 WT Probe
64279 Fluorophore-2 AGG CAC TAG ATT ATT TGA GTG GGA GTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64275 0.40 uM
64276 0.40 uM
64277 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.