Protocol 37335: Probe Assay - Cdh18<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

>chr15:23226569+23226719 151bp AGCATGAGGTTTCCAGTAAAGA TGACAGAACCATCACCTTTGTC

Mut = 154 bp

Wt = 151 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CAATGTTCACTTAATACCAATTAGAGAAAAAAAAAAGAAATGAAATGGTTGCTATAGCATGAGGTTTCCAGTAAAGAATAAAACTAAAATTCTTCATCCcttgataatattcaggttacaatgccatcagaatatattcatactgagttcctctatgtgtattttctagctgcactccaactccgacaaaggtgatggttctgtcaagtacatcctgactggagaaggggccgggactatatttatcattgatgacaccacaggtgacatccactcaaccaaaagcctggacagagagcagaagactcactatgtgctccatgcccaggctattgacagacggacaaacaagcctcttgaacctgagtctgagttcatcatcaaagtgcaagatattaatgacaatgccccaaagttcacagatggaccatacatcgttactgtgcctgaaatgtcagatatgggtaaggacacttatttttatagtgaagaaatccattcccattatttgtatcaaatatatcttttaaagaattactttaatgtattttttcaaacattgagaaagctctaaaatgttataatatttttattgcagtaagaacatctattattgaaccatacattatatattgaaaaaattcttaatatggctgtgtagaaatacttgtaatatctcaagccaaagccataaCTAGTTATCATTTGCAATTCTTATTTATTTGCATTATTCTTGAATATGCAAAATTTTTCATGTTATTA

This mutation is a 595 bp deletion beginning at Chromosome 15 position 23,226,613 bp and ending after 23,227,207 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51092 AGC ATG AGG TTT CCA GTA AAG A Common A
51093 TGA CAG AAC CAT CAC CTT TGT C Wild type Reverse A
51094 TTC AAG GCA GTT CCT GTT GA Mutant Reverse A
51095 Fluorophore-1 CAG GTT ACA ATG CCA TCA GAA TAT ATT C Quencher-1 WT Probe
51096 Fluorophore-2 CAT CCC TAG TTA TCA TTT GCA ATT CTT AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
51092 0.50 uM
51093 0.50 uM
51094 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 -- repeat steps 2-3 for 40 cycles
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.