Protocol 37355: Probe Assay - Ccdc40<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

>chr11:119234662+119234812 151bp CAGCGCATCCCTATCAAAGA GCAGTGGCATTGTTACCTGA

Mut = 151 bp

Wt = 151 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

AGAGAAGGTGTTTAGATGGGAGAAGAGAAAGTGGTCCCCCACAAGGCCCTTCCTGGAAGCGCAGAAAGTGAAGTCTAGCCCCTGTGAGGAGTGTGTTCAGGCTACTCTTTCCAGCCCCTGGCATTTGGGCAGCAGAGCGGAGGGTCTAACAGACCCATTCACAACCTTGATCAggaggggacacagttaaactcacatacaaactatagccgtgttcactcatttgttcctgacattctttacgtaggtgagagccccagcgcatccctatcaaagagacttcccaatgggggcacgacaccgattcagactgagcatcatgggaagtctgactccctcggacacggacgatcttcccctggaaacggacgagccgcctcagcaagagtcaggtaacaatgccactgcctgcgcccgcatgttatttacaaataacagggggggggggtgctggggagatggctcgggggttaagagcaccgactgctcttccaaaggtcctgagttcaattcccagcaaccacatagtggctcacaaccatctgttgtgggatctggcgccctcttctggtgcgtctgaagaaagcaagagtgtacttacataaaacaaatctgagaggggggggggggaagaaaactaggaggatccacctagcctcgtaAGAAATGGGACAGTGGTTCTGAGTGCAGCTCACTGGTGGGACTTGCTTGGCACACACAAGGCCCAGGCTCTGGTACACAGAAATGGATGCAACACTTCACACCAGAGTCCCAGCAGTGTGAAACCTGAAGCAGGAAGACCCTTGAG

This mutation is a 489 bp deletion beginning at Chromosome 11 position 119,234,578 bp and ending after 119,235,066 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51138 CAG CGC ATC CCT ATC AAA GA Wild type Forward A
51139 GCA GTG GCA TTG TTA CCT GA Wild type Reverse A
51140 CCC TGT GAG GAG TGT GTT CA Mutant Forward A
51141 GTG TGT GCC AAG CAA GTC C Mutant Reverse A
51142 Fluorophore-1 CTG AGC ATC ATG GGA AGT CTG ACT Quencher-1 WT Probe
51143 Fluorophore-2 ATC AAG AAA TGG GAC AGT GGT TCT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51138 0.40 uM
51139 0.40 uM
51140 0.40 uM
51141 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.