Protocol 47164: Probe Assay - Ppp1r11<em1(IMPC)J>-Alt1
Version 2.0

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=  99 bp

Wild Type =116 bp

>chr17:36950289-36950404 116bp AAATAGCTATACCATGTTGCA GGCCTTTGATCTCACTTTCT

 

Sequence

Wt Sequence (deletion in lower case):

TTGTTGTTGTTTTTGTTCCCGTGGATACAAGGGATCAAACTCAGGTCTTCATGCTTGCACCAAAAGAGCTTTACCAACAGAGATGTCTCCCCATCCCTGCTTTATTTTATTTTATTTTTTTCTGAGAAAATAGCTATACCATGTTGCAGAGGCCATTTCAgtctcccaagtgctgagattacatacctcagtctctttagaacatgaataagaggtgtgaagaagaaagtgagatcaaaggccagtcacaggaaagggagaaaataacattcatatattaatgccaccaagggtgatagcgttggttgtatcttagtttgccaatttgctggcaccttccctagtttaagaattgccttctttgcatactattggttatcaagaacagggattcatactctccacaaacaggctgtcctgcctctccaggggcatgatgtgtgttactgactgagccagggaagagaaaaaaggaggttccataggaaacaggaaaaatcaagaggaagtgagtcagatgggcccacacctcttttaacttttcaggagaatcagagcctaactatgaaacttcggaaacgcaagccagagaagaaggtggagtggtccagtgacactgtggacaatgagcatatggggcgacgctcgtcgaaatgtgagtgtgggtggcccagtgtccctggccaccatctcactgttgctttatagctctatcgagttcttggttggaaagaaaaataggtgggaataaaactatgcGGTACCAACTGCCTGGAGTTAGGGGTGGGGGCGGGGGCAAGCCATGGCAGAATGGGAGTCAGGTTTTTCCGACTCTCTCTTGAATTCCTGGGGGAAGAGGCTAAGGCAGCTGGATTCCAGAAAAGTAGGGAGAGGTTTTCACACAGATCTG

This mutation is a 599 bp deletion beginning at Chromosome 17 position 37,260,665 bp and ending after 37,261,263 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
68924 AAA CCT GAC TCC CAT TCT G Common A
68925 AAA TAG CTA TAC CAT GTT GCA Mutant Forward A
70005 GGC CTT TGA TCT CAC TTT CT Wild type Reverse A
70006 Fluorophore-1 TCT CCC AAG TGC TGA GAT TA Quencher-1 WT Probe
70007 Fluorophore-2 AGG CCA TTT CAG GTA CCA ACT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
68924 0.40 uM
68925 0.40 uM
70005 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.