Protocol 37483: Probe Assay - Trim69<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

>chr2:122174482+122174582 101bp GGGAAGGAGCTATTGAGTCCTA CTCCACCCAAGTCCATCTC

Mut = 103 bp

Wt = 101 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AAAAAAAAAAACCAAAAAAACAAAAACAAAAACAAACCAACAATTGAAAACAAAGCCTCTCATCCTACACCATTAAACTAGTTGGGAAAGAAGGAATGAACCTCCCAAAGGGCTTTGTGTACTAGGATTGAATGTCGATGCTAGGCAGCCATTTGTGTGaaattccccaaggtgtgtggtgtggtgtaagtccctctccccaaatgctagtctcagtagtaatcgtggtgatgattatggaggtttccaggacaacaagatacaactgcaacaaaacctttccttggagtttctaaagctacatcagtttctgcacaacaaagaaaaggacattctaaatgacctccgggatgaggggaaactcttgaatgaggagatggaggtgaatctgaaccagattcaggagcagtgtcttgttgccaaagacatgctggcgaccattcaggcacggatggaacagcagaactccttcgacttcctcacagtgagattctgaatccaattgattatggggacacttggaaggagggaagagaaactgggagtggggggtggaaggaaagcaaatcataaccaagctcaacccaacccaaccatgtcctaacctacccaacccaacccaatttaacccaacccaacccgactaactggagcgcttgtaggtccttggggagaaaaaggaagccatgtttactttatgatcagtggaaatttttactttcatcttccttcaaaacgtgataggacaatttcactaataatgctacatggatcagttctttcttacttccccatatagaccacagaagctttatttttcaaaaaatatgtctcttatagtgtttgtagagtttgggccttgcagaaattataaactgaaatatatctaaaaagctttttgtcaaacttttgctacctgaagtctcacttctagagatcagatgattggcgatcactccagcactcagaagtctagttacccttccccctcagatcgtttctgatcttttcttggcggagtatttggaagcactcacttctcaaagagttgatcttaactatgggcttcctggctcttctcttgtttcctatgagagctggaattatagaaaatcagaaggttaccatgtgagaggtcagtttatgctgggttagcctgggatgctgcttagcaaagtctgtagaggatgaaatgttgttggcgatggcctgaggaccatctgtacaggaagccagtattgatgatcaaacctggatggctttcagtataaccagagaagttcctagagatgatgaacaggcatctgacttctctatttcttcttttctcttctaggacatcacaaagctcatagagaggcaagtgtttcttcatggttctctctaccctccttcttctacccctggagaatgtctaaaactaaactgtttgtgtgtcccttacagcatggagaaaggaatgaagacaattgtacctagacagctcatcgccaaaaagctgagcctaggccgcttcaaaggtcctatccagtatatcatctggaaggaaatgcaggctattctctctccaggtatagtgggaaggagctattgagtcctatggcccctaatctatatttcatctgtggccagagttagttctgtagtttgagagAGGATGGAGATGGACTTGGGTGGAGTTTAGTCA

This mutation is a 1495 bp deletion beginning at Chromosome 2 position 122,173,063 bp and ending after 122,174,557 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51442 CTC CAC CCA AGT CCA TCT C Common A
51443 GGG AAG GAG CTA TTG AGT CCT A Wild type Forward A
51444 TTG GGA AAG AAG GAA TGA ACC Mutant Forward A
51445 Fluorophore-1 CAT CTG TGG CCA GAG TTA GTT CTG Quencher-1 WT Probe
51446 Fluorophore-2 AGG CAG CCA TTT GTG TGA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51442 0.40 uM
51443 0.40 uM
51444 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.