Protocol 36273: Probe Assay - Mphosph8<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

>chr14:56676330+56676447 118bp GTATTGGGTTTTGGGTTTCA CTACACACGCCTGAGCACAC

Mut= 119 bp

Wt= 118 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GAAGGGCTTTCACGGTGACATTTTCATACCTGTATATAATGAATTGCAAGGTTTTTTTTGTTGTTGTTGTTGTTTTATTTGAAATAAGGTCTCACTCCATGACTGAGCTGGCCTTGAATTTATGTAGATCTGTCCCTGCCATCCTAGTGCTAAGATTAAAGGTTTACACTATCATGTAAATGTATATTCAAACATTTTAAGTAATAGATTCTTAAAAGAAAATGGAGAAACAAAGTATTGGGTTTTGGGTTTCATTTCATATGGCTTAGCGTGTCTGCAGAATAGTCTTGGTggcgggagggagcagtgggagaaacattaaataatcagctgtgtgctcaggcgtgtgtagaatatttggtttgcaaatattacagtatcaactctttatcttttattttttgttttgcattcattattctttaggagaaagagaaaaaaaatgaacccaaaggaaaataccagaaaaggtatgatttggacaaagaagaaaaagccagaaaagagccaaaggtattaaaatgtaagtacaaaaattaaaatagttccatgttcattgccattgttaaaaaaattattaatggattttatgtgtattatgtgtacaccagGGTATATATATGTGCATATGTATATCTGATATGTTGTAAAGGTTGGATGTCTCTTTTCTGAAATATTTGGGACCAAAAATGTTTCTGATTCCTCTTCCTGGTTCAATGAAATTTATATGTACATATAATGAGATTTCTTAAGAATAGGCCCAAGTTTAAATCCAAATTTTGTTTTATATGTACATTATATGCACTCCTTTGTAATACTATGATGTTTTTAGTGTTCCAGTGTTTTTGAGTCTTGCATTAAGAGTAAAATTTTCTTCTTAATACCTTCATACTGGCTTAAGTTT

This mutation is a 319 bp deletion beginning at Chromosome 14 position 56,676,388 bp and ending after 56,676,706 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48262 GTA TTG GGT TTT GGG TTT CA Common A
48263 CTA CAC ACG CCT GAG CAC AC Wild type Reverse A
48264 TCA GAA AAG AGA CAT CCA ACC T Mutant Reverse A
48265 Fluorophore-1 AGC AGT GGG AGA AAC ATT AAA TAA TC Quencher-1 WT Probe
48266 Fluorophore-2 AGT CTT GGT GGT ATA TAT ATG TGC ATA TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48262 0.40 uM
48263 0.40 uM
48264 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.