Protocol 42459: Probe Assay - Ccdc87<em1(IMPC)J> Alt 3
Version 4.0

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

Mutant= 103 bp

Wild Type = 110 bp

 
chr19:4839608+4839717 110bp ACGCCCTTGCAATCCATC TCCTTGGCAGTTACTCGTG

The Mut For primer (primer 58286) anneals over the nucleotide sequence containing mouse genomic variations rs36883700.

Sequence

Wt Sequence (deletions in lower case):

GCTGTCCGGTAGTGCTACCGGCTGCGCAGGCGCATTGATGCAGTTTCCTTAGTAACCATTTGGCGTCCTCTACCCGCTCAGCCGGAACCACCTGGTAAAGATGGAACCACACAGTCAGGAACCTGACCTCAAGCCGATTTACcaccggttgctgagtccgctgtcgctcttccccagcaaggcgaagcctcctccagagcctccgaagcgcccctctcaagatgccacgcccttgcaatccatcccgctggcgaagctgaaagttgggccgctgtgccgccaagtgtccaagcggctagcaagcagcgggcgggcggcacgagtaactgccaaggaccgacttcgtctcactgaggttattctagaagagctgaagtgcaactggcgggaacctcctatagaacccattctggactacgaaaacaaccagaagctgcggcagcggctcgagtcctacgtgctgatcagtagtgagcagctcttcgtacgctacctgcacctgctggtgaccctgccgacctctagaagggtcttcactgaatcagccaccctcagccggttggcagtaaaccttgccagggattgcacagttttcctcaccagtcctgatgtctatcgctgtctgctggctgattttcagaccctgctaaatttaaagcacgcccagggaggcatagtcaaattgcgtcctcctccctgtccccctgggactttcaagctctgtccaatcccctggcctcacagcactggcttagaccacatgccgtgctctagcctcaacctgaactaccttgtccaactcagccgcccatatgatttccccagtgagcccgaacctgatccagtggaagagttgaaatccatcccccagctgaagagtagacagcagctcctctgggtgccctccatgataaaggataaagaaattgaatcgagaccctcaccgatggtaccactgcctagtcattctcccagcagtgagagtcaccaattccccacctcaccagtccattcctggctccaaaggggccagtccatgccttgtcttcatgaggggtggagtctggcagatgagttgtgccttcttcctccctctcctcatcccctcaccccgctgatcttggcttcagagagtaaaccactgccattccgggacatagtggctgaggatctgaagcagaaaatgaagatcatgagaatggagtggagtcgatactccctgctggactctggcctgcctcccctcctcggggtccttacccggcgcctaactgcccagcaccacctagagaaactgcagcaaatgatcaaaagcctgcaggaggaagaagcctccggaaagtgggatctccaacccccgagaatcatcccactacacccacagccagtcactgttgctttgaaggtacatgatcaagttatagtccaggtagctactgtgcaactctctgaaagatactttaacgactctttccacgttgagggggctggagtcctatacaaccatctgactggggaactggatggtaaagccattgaagaaatggatgcggatcgtcttgttgggaacaccaccggagaggtctacaaggagttgatgagccgagtctctgttagtcacttatcttttgaggaaggagaccaaattgagccttcagcagataaagattggtcctcctacttggcctcatcttttctacaccaagataaacatatgccaatcatcaaccgtaatctggttggattttactccagaagaacaagcactccgaagccagtccctgagaaggtgccctctctcacattactcccaaggcacaaaagctgggacaagcgaccaaacagacacggtgtatggatgaattggctgaaaccctcggtgtcttcagaggactacttcaagtacctctcctttcaggagtcggatttcctccacgtcatcttccaaatgtatgaaaaagaggctcctgtggaggtaccagtccctgtccaagagtacctagatattcagcaaccccctcccctgctgcaagatgaagagctagagttcatgcaaggaaaatgggattggagctcagtgatagaggatggctcaggacctgggagagcctacattcataacttgcagcagcgtctcaaacgactgtgggtcatgttggaagtccctgaacagaatcggctagacatggttattaaatacagctccaatgcacgcctgcaacagctaccagcattgatcaaggcttgggagcaggtcctgaagcccattcaaaagagggagtcgttgctaggaagactggagtggtttgagcaacaagcctctgatcccaatcgtttcttccaaaagcctgatttgttgatgaatcggttactggaggagaatcggttccgtagctatctccaaaggaagctcaatcgaatggaatctaatttggtttccttgttggaaaggatagagtcagtctttggggagccagtgaccttcaaggggcggagctatctgaagaagatgaagcaggacaaagtggagatgctgtactggctccagcagcagcggcggattcgcaatctgacccaggcccagaaaaccttccgccaatcatgcactttcacagggagcagcagccaggcgttagtggcccccgggaATACTCCCACTACTCACTAAGGCGGCCAAGTGCCCCCAAACCCTTCCTCACCCCCAAATGCTCACGTAGAACTCTCAATGACTTTAAAAGAGATTGAGATTCTTTTTCTTGATATTCTAAATAAAAGGATCCCCAAACTTGGTATTCCCATGTATGTCAA

 

This mutation is a 2506 bp deletion beginning at Chromosome 19 position 4,889,552 bp and ending after 4,892,057 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58286 GAT GGA ACC ACA CAG TCA GG Mutant Forward A
58288 CAT TTG GGG GTG AGG AAG Mutant Reverse A
60431 Fluorophore-1 AAG CCG ATT TAC ATA CTC CCA CTA C Quencher-1 MUT Probe
60744 ACG CCC TTG CAA TCC ATC Wild type Forward A
60745 TCC TTG GCA GTT ACT CGT G Wild type Reverse A
60746 Fluorophore-2 AAG CTG AAA GTT GGG CCG C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58286 0.40 uM
58288 0.40 uM
60744 0.40 uM
60745 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.