Protocol 37815: Probe Assay - 1700020N01Rik<em1(IMPC)J> Alternate 1
Version 2.0

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

>chr10:21593727+21593821 95bp CCAGTACAGATGCCAATAGGG TCCTTCATCCTCCACATCAAC

Mut = 105 bp

Wt = 95 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CTCTCAGATAACCAACAACTAGTATGGACATGACTGGACTTGAAGCTGAGACTTCTCTGGGCTTTGTAATGTCTGCAGTCGGCTTTCCCTAAAGCCTCATGCAAACATGGCTGATACTCCGGGGTTTACAACAcactgcggcaggggaggggcgggacttccagggcggaattttcggtgacatcaccacagaaagttcgttaaagcaagggctttggctgcgttctggaagtagggcccgaggtctgtcggtggggtggcttctgcagcgtgtggccgtgtgaccccacggaggtgacctcccaggtgagtcctccgcgtccctcggtggtgatcagtgtgtcgtcctgagatgtagtgtcgtggatacccagtgtgccctcttctctcgtctcttccacagagcggctgaggttgagaacccattgagcagctaggccccagtgtatgcgctgatcctatggcggaggcccgtgggaaccaaggcgagagctgtgtgagcctggaggacgtgtgcctgcgtttcactgcagaggagtggggtctcctcgatgacactcagaagctcctgtaccacaacgtgatgctggagaatttcttgcttgtgctgtccctgggacttccgatttccaaatccttcccaggtaccccagcgatagatgctgtcttagtcagggtttctattcctgcacaaacatcatgaccaagaagcagttggggaggaaagggtttattcagcttacccttcaatactttctctgtgataactccagctgtgtcaagttgacacaaacccagccagtacagatgccaatagggactccctgttaaccaaagcatgatggcgtaggtaatctaagctcgcTCCCGAGGTTGATGTGGAGGATGAAGGATGTGAGCAGAGCATCTTCAACAAATGCTTCACACCCTGAAGCTTAGCTCTGTAAGAGTGTTAGGCATCAAATAAAAAATTTAACCAGTTATCCAGCCTGGAGAACCCAC

This mutation is a 732 bp deletion beginning at Chromosome 10 position 21,593,062 bp and ending after 21,593,793 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50572 TCC TTC ATC CTC CAC ATC AAC Common A
50573 CCA GTA CAG ATG CCA ATA GGG Wild type Forward A
50574 CTG GGC TTT GTA ATG TCT GC Mutant Forward A
50575 Fluorophore-1 TCC CTG TTA ACC AAA GCA TGA TG Quencher-1 WT Probe
52220 Fluorophore-2 CTT TCC CTA AAG CCT CAT GCA AAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50572 0.40 uM
50573 0.40 uM
50574 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.