Protocol 33050: Probe Assay - Pgm1<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

>chr4:99961303+99961413 111bp CTCAGTAACATGGTAGAGATATCCAAG GAAGAAATGCCAGGGGAAGA

Mut= 129 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AGTCAGTCCACATTGAGAACCTTCCCCAGATGGACTGTCACACATGCACTGTGTTCTTGCCTTACAGCACCTCAGTGTATGGTTCCAAAGCTACAAAGTTCCATAGGGAAGAAGGGAAAGGAGGGAGGGCGGGCTTTGCTCAACAGCTCTGTTCTGTCTCAGTAACATGGTAGAGATATCCAAGATGaagatgtttctttaagaaagcatctttgctggtccaacagtgtcttcattgtccatgttcttcttcccctggcatttcttcgtgggagctttgtatttatatgttctggatttcctatcctagattggtcgcctggttattgggcagaatggaatactctccacccctgccgtatcttgcatcatccgaaaaatcaaagctattggtgggatcattctgacagccagccataatccaggagggcccaatggagattttggaattaaattcaatatttctaatgggggtgagtattctgtcatcttaaggacaagtagatacagttggataagctagtgcccgaagccctgggctctgattcccatcttaagaaggggttttgcttccctttggagatggtaaagtacttcttagtctacagctgccttctttgtaatgaattgtagctatcatggtattctactttagaaaagtctaactggtattattagctcctagcactttgaaggttgacacaggagAATTGTGAATTCAAGGCCAATCCAAACTGTATAGTGAGAATTTGACTCAGAAACATCTGGAAAAAAAAAAAAAAAAAAAAGAGTCCTGGTGCTTTGTGACTCAGTACTGGGGTCGAGGGATTGCTGAGTGCTGGGTTGTGTTTTCTCATGAACCGCA

This mutation is a 521 bp deletion beginning at Chromosome 4 position 99,961,333 bp and ending after 99,961,853 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40551 CTC AGT AAC ATG GTA GAG ATA TCC AAG Common A
40552 GAA GAA ATG CCA GGG GAA GA Wild type Reverse A
40553 TCA CAA AGC ACC AGG ACT CT Mutant Reverse A
40554 Fluorophore-1 AAC AGT GTC TTC ATT GTC CAT GTT Quencher-1 WT Probe
40555 Fluorophore-2 ATG AAT TGT GAA TTC AAG GCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40551 0.40 uM
40552 0.40 uM
40553 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.