Protocol 34612: Probe Assay - Prr5<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

>chr15:84693392+84693500 109bp CTTCCCCGGATGTTTCTAGC AGTGAAGAAGGACCCCAGCTC

Mut= 115 bp

Wt= 109 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (A insertion):

 GTTTGGTCGCATTGGGACCCTCAGACTTCTCTTCTGTGAGATGCATAGTGAGCCAGCCACTCTGTGGGTCACAGAGGGAAAATAATCTCTTGGGCCACTTGTATTGCTGTGCTGAGCAGGTTGGTCCCTGGCTTCCCCGGATGTTTCTAGCCCTCACCAG^tactgtaggatctcaacttgtccccatctgccctcccccacaggcagctgctgaagacggagctggggtccttcttcactgagtacctgcaggtacgtgggccttgccacagaggaaggcttgctctgtctctcaaaagaggtctctatagttctgtgacctgtgacagcccgggcaggctctaggctggcctctcggggtgggtggggttttgactgcttggcaggagggtgggtctcatgtcttggctgcttgttggcgggctcctggtgggtcctgtaagccaaaccctcctgaaagagctacaggtcagtgatggcgcctctgttggctttcagaaccagctgctcacgaagggcatggtgattcttcgtgacaagattcgcttctacgaaggtgagtcttactcggagctgagtgtggccagcctggctgtgatgtggtgtggttgatgttctgggacagtggcgtggcagatgct^GGGAGGCAACGGCGGCTCTCTGGTTAAGATGGCCGGAAAGGAAGGGAACAGCATTGGGCAGCGGGACTGAGGTCTGGACTGGGAGGGGACCTTGGAGAGAGTCTGCACCCACCCCCTTGGGAGCCCAGGAGGGCTGCGCAACCTAGGGAG

This mutation is a 481 bp deletion beginning at Chromosome 15 position 84,693,421 bp and ending after 84,693,901 bp (GRCm38/mm10). There is also a single bp (A) insertion at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44021 CTT CCC CGG ATG TTT CTA GC Common A
44022 AGT GAA GAA GGA CCC CAG CTC Wild type Reverse A
44023 CTC CCA GTC CAG ACC TCA GT Mutant Reverse A
44024 Fluorophore-1 TGT AGG ATC TCA ACT TGT CCC C Quencher-1 WT Probe
44025 Fluorophore-2 AGA GGG AGG CAA CGG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44021 0.40 uM
44022 0.40 uM
44023 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.