Protocol 31717: Probe Assay - Prmt9<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

>chr8:77555755+77555864 110bp TTGGAGCTGTTTCCTGATGA CTTGCTTTGGCCTGATAGAGA

Mutant=  129 bp

Wild Type = 110 bp

Sequence

Wt Sequence:ACTGCTGGACTGTCTCTGTAGCCCCTTAGTTGTTTTTATTTATTTGCTTATTTTTGCTGTTACCatgagccaaacccaggaccttaagcatcctaggaagtcctcattcacgtttgttgaacagtacttactaaaggttaccagttggaggccatcattcatttcaggctcagttccacttgctggagtcttcagctagagttgcgtattcctcattcttttttactaatttgtacttttttcttgtttataaaggaaacttttcagtatacacttttcaagtgggctgaagagcttcatgcactgagccgaatacaagacttacttggttgctatgagcaggccttggagctgtttcctgatgatgaagtgatttgcaacagtatgggagagcacctcttcaggtttgtcatgacctcATGAGTATAGGATTATCTCTATCAGGCCAAAGCAAGGATAAATCATGCATTAACTTACTTGGTTTGGTTTTGAGATAGAGTCTTACCACCAGCTCAGTCCAGTCTGGAACTTGTAGGCTTACATCTTGTTTTAG

 

Deleted Sequence: atgagccaaacccaggaccttaagcatcctaggaagtcctcattcacgtttgttgaacagtacttactaaaggttaccagttggaggccatcattcatttcaggctcagttccacttgctggagtcttcagctagagttgcgtattcctcattcttttttactaatttgtacttttttcttgtttataaaggaaacttttcagtatacacttttcaagtgggctgaagagcttcatgcactgagccgaatacaagacttacttggttgctatgagcaggccttggagctgtttcctgatgatgaagtgatttgcaacagtatgggagagcacctcttcaggtttgtcatgacctc

355 bp deletion beginning at Chromosome 8 positive strand position 77,555,474 bp and ending after 77,555,828 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37473 TTG GAG CTG TTT CCT GAT GA Wild type Forward A
37474 CTT GCT TTG GCC TGA TAG AGA Common A
37475 ACT GAA CAC CAA GAG CAG CA Mutant Forward A
37476 Fluorophore-1 TGC AAC AGT ATG GGA GAG CA Quencher-1 WT Probe
37477 Fluorophore-2 CTC TTA ACT GCT GGA CTG TCT CTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37473 0.40 uM
37474 0.40 uM
37475 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.