Protocol 34275: Probe Assay - Gm5547<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

>chr3:105815164+105815302 139bp TCTGGACTCTGGACATTCTACA TTGTTCACACCTTGTCACTGAT

Mut= 134 bp

Wt= 139 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GATGTGAACTCATTTATGTTGAATGGTCTAGATGATGATAATGCTTTGGGCTTGTTTCTTAGAGTGTAGTCTGGACTCTGGACATTCTACAAAATTTAATTTCGAGTATAAGCAGAGCATAATGACGACCACCCCATAAGAatttggatagaaggacggcagcaagtgtgggtcgggggagccttgatcagtgacaaggtgtgaacaaggcagaggctttgggctgacaaggaacagtcgccagacttcaaagcttgtgatgaggctgacagcattgaggagctgtgtagagacttggctgtcagccctcctgatagtcacagatagggcagctttcatgcagagttctgtacacaccctgggctgccttcagggctgcatggatgtgggtagagtcatctcagcagccgcaggctcctcctgctatacccacttcctcgtttgcaaactgcttcacttctgagagaccagggccctttccagtagctttagggatggtaagttgtaagtgattcttgctaagtattccttttatgctcttaagtctcatcagagagataagaaatcagggcccagataaacaggtgcctgtgttacattcactgtaagtgagtctgcagtgaagctcacgtgaacatgctggaggcaaaggcaagttccagggttcaagttcagtaggattctaacagcaaacaatcacgccgctctgcttcttttgtttaaaacaaacaactagaaaatgcctccttattaggtaggtactgtacgctatgattgttaaatcgccatcctgaacatttcaactgtttgtgtccccatgcagctgtccatgacccaaacttggaggaggacaagtgaatgcatctcataaaggagaaggtaatataatcaatcgctttatctaacatttctacctgggcaaagatggaagtttaattttcaaggtacacggatactctctgttgacactctaccacacggcagaatgctctgctgatctcctctgccctgagttaatttgggacctttcctctcagagaaacagtaaacaatattaattttcacagcattgtgaagttggtgacgtcgctcaagcatgatgacagctggtcagtaggtcacaagttctcctcgttgttattcccacagactgggaagccaaggcggagaaggacatgtgagactgggcaggcttcagctgtcatctgtgggaagtggcttttggttgaccagcggtacttcccagtgtcagcacgtgtgttgtgtcggcagctcagaccttgcgctgaggggcccctgtcctcagccacccttcatcttgggaaaactctctggtgtagagcagggaggtagggaggcagggaggcagggaggcagggaggcagggaggcagggaggcttcctagtgctaactcaacttgtttaaaccattatactttatctaaataattgcattgaatacttcgatacaagtaagaacacgcaggagagaagtaggagaggctgccgtggaagaagaagaatatgcaggagagagaaggagagctgccatgtgctaaagggccaagagaacgtggcccagagggctgctcagttgggtcaagagcagccaagatggaacatagaaactagtaagtagtaactcagagttaacagtaggaaagtggattctaacagcatagagagtaggcagttacccagctatttgtgctgcctaaggcatatatatatatatatatatatatactttgatacaaagccctactatgtctgtgttgttatccctgttttaaaagaggactccatcgggttctcgtagttaggaagctatgaagtcaaactttaaaacccaagggttcctgactgcaaagtccactttctttccagttgccttgctgaagtgcccgctctgaatgaagaagaactgggtcctgcagtctgtccacaggaagaagactgtctgtgagcacagtctccatgttcactgtctcatgccgtccctgatatcatggaggaaatgaatggtcatttaagaatcctcattggctaaagagctggtggtttaaatgaatgaatctaatgcaaactgtgaagggctggtcctggtattaagaagatggacgattctgtgaagggaatgtgctaagacttgtttgttgaaaatggaattattaatggcttgtcagtaaagttaagaatgccttaaatcattcctctcttactctattcatagaagaaataaaacagatgaatttataaagggtttattgtctgccagacattattttttatatttatatgggtcatttaattctgcaatgatcctacttggaagatatagttatttattacagttgaaagttcgggggaagaacagaaaggttaagaaacttgcccaaggatacaaaggatggaaatggtaatggggcagcctgaacttccaaatctggacagctattcactgtcccttctgctcctgtctaatagaagggaagatcagacagaggtatgaaggtctgtgtctatatttagaaaaaaaattcttctttctcatttttttattcctagagggatagccactttttgcaatgctgagccatacTAATGGCAAGACAAGTCTACTGACTATCACTCTTAAGTCTCTCACCACATATCCTCATCCCTCTCTTTCCTTCTCAGCATGACTCTAGCATCCTCCTCTGCCCTTGGGAGTGAGGGGTGACTGTGGACTAA

This mutation is a 2461 bp deletion beginning at Chromosome 3 position 105,815,236 bp and ending after 105,817,696 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43201 TCT GGA CTC TGG ACA TTC TAC A Common A
43202 TTG TTC ACA CCT TGT CAC TGA T Wild type Reverse A
43203 AGG GAT GAG GAT ATG TGG TGA Mutant Reverse A
43204 Fluorophore-1 AAG GAC GGC AGC AAG TGT Quencher-1 WT Probe
43205 Fluorophore-2 CCC ATA AGA TAA TGG CAA GAC AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43201 0.40 uM
43202 0.40 uM
43203 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.