Protocol 34919: Probe Assay - Pole3<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

>chr4:62524396-62524487 92bp ATGCCTCCACCCACTCTGAC TGAGAGTCTTGCGCTTTCCT

Mutant=  109 bp

Wild Type = 92 bp

Sequence

Wt Sequence deletions in lower case;  insertions with carrots ^ct^ :

GCAGCTCCCGGACGGTGTCAACATCTCCAAGGAGGCCCGAAGCGCCATCTCCCGCGCCGCCAGTGTCTTCGTTTTGTATGCCACATCCTGGTGAGCTGAGCAGAGGCTGAAGGGAGGCGCAGCGGGATCCTGACCCCGCGTTCGTGGCCTGGAACCTACAGGCCCCAGTTCCCTGCCTCAGTAGAGAGCGACTTACGGCTCTCTTCCAGTTGTCACACATTCTGGAGCAGGTTAAACAAGACGGTTACCGTAGCCCTTTGTATGGGAAGGGGATGAACTGAGCCATGCCACACACACACACAATCCATTCATCTACGATAAATACGGCTTTCCCACCCCATTCCTGGGGGTCATGCCTCCACCCACTCTGACAGCTTCT^ct^gaagggcgttgctctttcttccagtgccaataacttcgcaatgaaaggaaagcgcaagactctcaatgccagtgacgtgctgtcagccatggaagagatggaattccagcgattcataacaccattgaaagaagctctagaaggtatccacgtactcttctgtctcttaaaccatatataaaaaaaatgctcatcagtatcagtgccattaagtcccgttcaataatacaggactggtcataactcccctctgtttagttggtaccatctcgggtatgtgataggggcctggttattccacctgtgcctcggtttgtctaattaaaccataatatcctgcttccttacagcgtacaggcgggagcagaaaggcaagaaggaggcttcggagcaaaagaagaaggacaaagacaaaaaggattccgaagagcaagacaaaagcagagaggaggaggaggaggatgaagagagactggacgaggacgaccagaacgaagaggaggaaatagacaactgaatggagagagtaggcagactgtggcgtggtggaggggaccctgagatcctgagacatgtctgtgtgaaggctttccctgcaaggcagagccatcttcggcactggtgcctgagatcaaaataaaagctgaccagaaagatcaaaaagaactcttcccaggctcagagcatctgagttacagaaaccggtcatgcgtcatcagtctgcaggttaggatgtttttgaataagcgattctgagtgactacatagtttggtattgcttgtctacttaaagtatatggctagctagtttctgatcccatttccctgcccctctgaataagtaataacatccatgttgcctgcagtgttccaagtcacagaagcactaaaggctcagggggaaacaatggggctttgagcctggtcacccagtttgactgtacagtagttgaccataggaactcgtgcttagaatggctgctctctaggagatgactaaggagctgtgagcgagcgaacgaggaaatgtcttcacctgatggtagcattacttacttggtgtctggctttctagacttacttagctttgtagtcctctagtgactgtagatgctcagtcgtctggggtggctgcactcataggacacacactctttaattctgagtgttgattagaatctgtcaaaaaaggtgccaattcttaaaagcaggggcttaatcttggaggtaactcagtgcctgccatggtaggtcctagtgctacctcctggaaggtataaaatcccagatccatctcccagacttatggctgctgcttttcattccctgggctcataacaagttccagagactttatttcttccaggcacagcctgttctgtctccatctctctggcttctgcttcccagcccggctttctcctcctcttacttatttcatcttctagtctagggactctgataacatatgtcatattccaaagtgcaaggtcctcagattccttagtcctaatctcctgccttctaaagagagttgagaacacctctgaaccttacggtaagaggaatggtgctcaggaatgtagcaccactgctgtggcttcgaacgtgtctgttggcctttctttttccccattgagtgctccaagcactttttcctgtcttagttgtgcacttgaggctgacaggtgatgtaatatagttggacactgtttactagtaatacagggaagagtttggtgagagtgactggagactgttgagaagattttaatgctttctacaaataaagaacattcttgtttaaaatgatgactcatacaatgtgcttttgCAAAGGACTCACCAAGACCCCCCTCTCTCTCCCCCTACCCTCACACATAGGCACACAGTCTTGTGTCTTGCATTACCAGCAGGTGAATGAGTATCACAAGTTAAGGGCACTGTTCTTAACCACAGCCAACATATCCTGAGATTCTCCTGGTTACTTTGCATATCTCACTAGCTCCCACTTCTGTTTGTTTGTTTGTTTTGGTTTTTTGAGACAGGGTTTCTCTCTGTAGCCCTGGCTGTCCTGGAACTCACTCTGTAGATCAGGCTGGCCTCGAATTCAGAAATTCACCTGCCTCTGCCTTCCAAATGCTGGGATTAAAGGTGTGTG

A 1834 bp deletion beginning at Chromosome 4 position 62,522,627 bp and ending after 62,524,460 bp (GRCm38/mm10). In addition, there is a 2 bp (CT) insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44803 ATG CCT CCA CCC ACT CTG AC Common A
44804 TGA GAG TCT TGC GCT TTC CT Wild type Reverse A
44805 GCT GGT AAT GCA AGA CAC AAG A Mutant Reverse A
44806 Fluorophore-1 CCT ACC CTC ACA CAT AGG CAC Quencher-1 MUT Probe
44807 Fluorophore-2 AGG GCG TTG CTC TTT CTT C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44803 0.40 uM
44804 0.40 uM
44805 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.