Protocol 34608: Probe Assay - Mphosph10<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

>chr7:64389119-64389220 102bp CAGCCTTTGCTAAGTTGTGC ACATCACCACTACCCCACCA

Mut= 100 bp

Wt= 102 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

ATACTCTATTTTACCTGTAGGTCTCAATCCCATTTCAGATATCATATCCTTAGATAATTTTTCTCAAGCTTAAAGCCAGGTGGGAATCACTTGCCAAACGACTAATAGGTGAGGAGTATGTATGATGCAGATTTGAGGATTTAAGGCTGACTTCCTGGgtatctccaggcaagtagatgcgtaagcttcagtatatacagcaaagtcaggtttttagtttttacggtctggctcatcagcagggtactgttgggaactagagtctgtacttaaaattgatgcttcatttacattttaatttgcaaatacgatatgtctttttcttttatttaaaaatgcttgatgctattgtgtataattcattctcacttaattttctttccaatagaattcagaatggactagcatcaaacttcacatctttaacaaaagttctatatgattttaataaagtattagagaatggccgaatctctggaagtcctttacagaaacttgagataaacagttttgatgatgagcagatttggcaacaactcgaattgcaaaatgaacctgtcttacaatacttccagaatgcagttagtgagacagttgaggatgaagatatcagccttctcccagaatgtgaagatgaggagtgcgaagaggatgcctcagaggtggaggctgacaaccaggagaacctagagacagacttggatgaagaacaactgtcagacgagggtggtgatgttcctaaagggagggacagagccaaaagctccaggaaatctgacccaaggaaaagtccagttttcagtgatgaagattctgaccttgactttgatattggcaaattggagcagcagaccaagatgcaaatcaagccacctggaaaaccaagagaaaagtctgtcgtagatgataagttcttcaaactgtctgaaatggagagcttcttagaaaaagtagaaaaagaagaggaaaaaagaccagatggtgaagaggaggacgaggaagacattgacctttttgaagacattgattcagatgaaagcgagggagggctgtttgggcggcagaagattaaggtaatgttttggaaaaaaacaaaaactgtttctttaaagtacaagtcatgttttcctacagagaaatgtaatgtgcctcagacctcatttgagcccaaatgaagtaaggaaaaaacaaacaaacataaagaaataaattaaaattaagagctgagccagtggtgcaggagtgtagtaatcctcagaagtgggaggctgaggcaggaagattgtggcttctcaaagccagcctttgctaagttgtgcttcagttgtccttatggttttatggtgGTCAACTAGTTGCCAGTTTCCCTAATAACTATTTCTGGTGGGGTAGTGGTGATGTTAGGAACCAGTTTGTGTGTGTAATATACATAGGCACATGAGTATGTTTATATATTTTTTTATCTTTAAACTTTTCAAAAATGTTTTTTATTTTAGTCAAATAAA

This mutation is a 1188 bp deletion beginning at Chromosome 7 position 64,389,174 bp and ending after 64,390,361 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44000 CAG CCT TTG CTA AGT TGT GC Wild type Forward A
44001 ACA TCA CCA CTA CCC CAC CA Common A
44002 GAG TAT GTA TGA TGC AGA TTT G Mutant Forward A
44003 Fluorophore-1 CAG TTG TCC TTA TGG TTT TAT GGT G Quencher-1 WT Probe
44004 Fluorophore-2 AGG CTG ACT TCC TGG GTC A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44000 0.40 uM
44001 0.40 uM
44002 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.