Protocol 35961: Probe Assay - Arglu1<em1(IMPC)J>-Alternate 4
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

>chr8:8683447-8683604 158bp ATTTTTTAGCCAGAAGTGCTGA CCCCTACCCTCCCAAAAA

 

Mut=  124 bp

Wt=  158 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case, insertion in ^g^ (ACCTG Insertion):

 GATTTTTATCTCCATTTGAATTTCTGTATTACTTATCCTGGCTGCAGTTTTTTATCCATTCATTTATTTATTTATTTTAGTGTGGTGTGAAAACAAGCTATATAATTATTAAGGCTATAAGAAATGAGACTCAGTTTAGTAGTAGGTTTAAAGTTAGTT^ttctaattcttaggttctttaaaatattaagttgtcttttctacttttatttgctagtcggcagcaggaaatagaagaaaaactcattgaggaagaaacagcacgaagagtggaagaattggtagcaaaaagggtagaagaagaattggagaaaaggaaggatgaaattgagcgagaagttctccgaagggttgaagaagccaaacgcatcatggaaaagcagttgctcgaagaactcgagcgacagagacaagctgagcttgcagcacaaaaagccagagaggtaacgctcggtcgtttggaaagtagagacagtccatggcaaaactttcagtgtcaggtgtgcctcctgctcagtccagaacgagatggaatgcgactatcttaattcctttctcatctaaacttacatggctgcgaaagataattttttagccagaagtgctgactggtacttaaaagttactttcttaaaacttattcaaggatatttttgatccgatggaactggcttacatatttgagaagtgtttgaaacttttgc^CATGGCTGCAGGACTACATTCTTTTTTGGGAGGGTAGGGGGAACCAGGGAGTGGTAAAGGGAAAAGGCTAAAGATCCACCCGCGGTTGCATTTTCTTCTCTGTTATCACTCTGCTACCTAGACTGTGAGAGGCTTTGCCTTCAGTCAGATTACAGAGAGCAGGGT

The mutation is a 544 bp deletion beginning at Chromosome 8 position 8,683,487 bp and ending after 8,684,030 bp (GRCm38/mm10). In addition, there is a 5 bp insertion (ACCTG) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45186 Fluorophore-1 CTG GCT TAC ATA TTT GAG AAG TGT Quencher-1 WT Probe
45187 Fluorophore-2 AGG TTT AAA GTT AGT TAC CTG CAT GGC Quencher-2 MUT Probe
47420 ATT TTT TAG CCA GAA GTG CTG A Wild type Forward A
47423 CCC CTA CCC TCC CAA AAA Common A
47424 TGT GGT GTG AAA ACA AGC TAT Mutant Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47420 0.40 uM
47423 0.40 uM
47424 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.