Protocol 34481: Probe Assay - Fam53c<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

>chr18:34769017+34769115 99bp CCTGGGAACTCTTCTGTCTAGAGT CTAAGGACTAACTTGACAGGGCAGA

Mut= 90 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTAGACCAGGCTAGCCTCGAACTCAGAGATCCACCTGCCTCTGCCTCCCAGGTGCCAGGATTTAGAGGCATGGCATTACCACTTCACAGCTCATACAGGTTTCTTGATTGACTGCATTTTTGAggaggagtggcagacagtgtataaacattcttggtgccaggtgggttctcaggctgcaggtagcatggtgtgggtgggtttagatcctgttctgttcccttaatagctgtgcctgttaaccttggacaagcacagtcatagtacttccttcatggagacttttgagaaattacattctgtggtacagtagctcttagaatatgttagccattaacccttttcattatgcatgcctttcagaaggtgcttcctggaggggcctgccccactgttcctgtgctgagttccaggacagcctcaacttcagctaccatccctcaggcctaagcctgcacctcagaccacccagtcggggaaactcccccaaggagccgcccctctcccaagtactgagccccgaacccccagacccagagaagcttcctgtgccccctgcccctccatccaagaggcactgccgctcactctcggtgcctgtggacctgtctcgctggcagccggtgtggcggcctgccccctccaagctgtggactcccatcaagcaccggggcaatgctggagggggtgggccgcaggtgcctcagcagagccctccgaagcgggtctccagcctcaggttcctccaagctcccagtgcctcgtctcagtgtgccccagcccacagaccctacagccctcctttcttcagcctggccctggcccaagattccgctcagccctgcgccacctcccctcagagtggctcctgggagagtgatgcagagtccctgtcgccctgcccaccccagcgccgcttctccctgtcacccagcctgggcccacaggcgagccgcttcctgccctctgcccggagctctcccgcgtcttccccagagctgccctggagaccccgaggtctccgaaaccttccccgaagccgctcacagccttgtgatctggatgcccgcaaaactggtgtcaagcggcgtcatgaggaagactgcaggcgccttcggccttccttggactttgacaagatgaatcaggtgggactggcagaccagaagctgtttaaacatctcctaattccctaccttctggcaaggcctccctgggaactcttctgtctagagtttatacaagaaaaccaccctggcagtattgaattttaaCCTTTAATGTCTTCTGCCCTGTCAAGTTAGTCCTTAGTATTCTAAAGAAACTAGACTGAGGCCTTAAGCCTTCAGACAGGGATCCTAATTGTCCTGCCAGAGGCACTTTTCCTCTTCTCTGAGAACTCTAGACCACAGG

This mutation is a 1151 bp deletion beginning at Chromosome 18 position 34,767,928 bp and ending after 34,769,078 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43728 CCT GGG AAC TCT TCT GTC TAG AGT Wild type Forward A
43729 CTA AGG ACT AAC TTG ACA GGG CAG A Common A
43730 TGG CAT TAC CAC TTC ACA GC Mutant Forward A
43731 Fluorophore-1 ACA AGA AAA CCA CCC TGG C Quencher-1 WT Probe
43732 Fluorophore-2 CTT GAT TGA CTG CAT TTT TGA CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43728 0.40 uM
43729 0.40 uM
43730 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.