Protocol 36415: Probe Assay - Papolg<em1(IMPC)J>Alternate1
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

>chr11:23885671-23885777 107bp TGGCTGAGACTTTTACTGGAAAG TTTGCCAATCCTGTTGAAGA

Mut= 100 bp

Wt= 107 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGAAGAAGAAGAAGAAATATCTAGTAAGCACTTGGAATTGGCCCTGGAAAGAGGTAGAAGTAACTTTTTAATTATGATACAATTAAAGAAATAAAAGTGAAATCTTTCCATTTGTTCTTATAAAATGCTCCCATCAAGAATGACCTCACTCTGAAGAAGGCTAAAAAGCATGTAACAAAAACtaaagagaagacggggattggctgagacttttactggaaagagctgcagttcattaatttgatagttgagttccttaaaggggccttaaagtgcttttacacatctcttcaacaggattggcaaatgtacagtgtatcagttgtgcagtaaaagactggaaacttgtacatgttctttatgcaaagtaacttaaatgattgaatgccttttgctttattttagaatctcccaccttctgttgtggctactgttggtggtaaaattttcacatttggatcttacaggcttggagtacacaccaaaggtaattgctttttagttatgttctagtgataagaatttttaggaaactgttttacttgtttacaattagcgagtactgaacatagttcccatatcttactacTGTTAATAAACACTGTCTTATACTCATCTTTCATACCTAGTTCTGCATTTATGTTTATGTAAACTTTAATGAAAATATAATTTATCATTCTTAAGAAATAATTTTATTTTTTTAGGAGCTGATATTGATGCACTTTGTGTTGCCCCAAGACATGTAGAGAGATCAGATTTTTTTCAGTCCTTTTTTG

This mutation is a 407 bp deletion beginning at Chromosome 11 position 23,885,389 bp and ending after 23,885,795 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47356 TCC CAT CAA GAA TGA CCT CAC Mutant Forward A Mut F
47358 GCA GAA CTA GGT ATG AAA GAT GAG T Mutant Reverse A Mut R
48713 TGG CTG AGA CTT TTA CTG GAA AG Wild type Forward A Wt F
48714 TTT GCC AAT CCT GTT GAA GA Wild type Reverse A Wt R
48715 Fluorophore-1 TTG AGT TCC TTA AAG GGG CC Quencher-1 WT Probe
48716 Fluorophore-2 CTG AAG AAG GCT AAA AAG CAT GTA AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47356 0.40 uM
47358 0.40 uM
48713 0.40 uM
48714 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.