Protocol 35507: Probe Assay - Cep68<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

>chr11:20240714-20240833 120bp CCTTAGACCAACTTCGTTTTCA GGAAAGGGCACTGTGTTGA

Mut= 119 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGCTTCATTACAGGAGATTTCCGAAATAGGATGAGTCTCGGTACTAAGCACTAGCAGCGGGCACCCTGAAATCCCCTGAACGCTCTGCTCGTCTTCCTTTTCTTAGTATACCAAGCAGTCGGTTTAACCGTAGGATCCTTAGACCAACTTCGTTTTCATTGATAATCTTCCCTATTCCCCCCCCCCCCCCagtcttgtgaagaaaactgttcttagaaacaagaagtctgcttggtttcaacacagtgccctttcccccttgttattgcagatggaggaagacaggcttcctgcctcccaggacctacttccagctcttcaagtctttggaactataactgtttgctcaggacaggaggctgacagtgaagacttccaggctacgcttgatccatctcaggtgttgggcctcagccagcaaccacacacctcaggtctccctctcccgccacagtggaagtccacggtgagcccaggtgctcctcagctttctagcagaagcatctctgcctcctcggtgggcagcagtcttcaggatcaccaagagaaggcaggacctcagagggcctcttttgccaatgtctcctctcccgagttgactgtaccccaggcagcccactctgtggtgggggcagggcctccgctccaggggtcagcacagcctctgacctctggcagtgatgctacaggcctgggtaagagacacctctcctttcaggcagaatactgggcctgtgcgctgccaaattctctgcctccttcccctaaccgccactccgcactctgggacccaaataaagagtatgaagatctgcttgactacacttacccactgaggcctgggcctcagctcccaaagcaacctgaaagtcatgtcctgactgagcctgttctgcaggactcaggtgtagatctggacagtttgtctgtctccccagcaagtactctaaagtcacccactaatgtctcccacaattgctcatcagcagaagtgcctactctgccattttctggagccagagagtcatgtcttaagcgctggcccttgggaatattccagaaacagggtggcacaagcttgtcatcctggaaccagcttgcatcaacccctagagccccaggcactgaagatgcttcttgggagaacagagaggcagccctgaggggcacagcggaggactgcctccctataggtgaggacctccgaatgggctctccccagctgaagacgaaggagaaagagccaccctttcccagacagaagagaggcaggcagcatgttagctgcccggcctgtgtgacgcctggatggccatcagaagaggaggtgggaagtgatgaggagtaccttgctctgcccaccaggctgactcaggtctctagtttggtgtcctactcaggtgccaggccctcctttgtgaacctacacactggggctgctgaggaacacagttcactacaagtctcagacagtgacaagccagcctcccccacattggactccagccacaggaagcatccttctggaacttcctttcaagggcctgtgggccagaacccctgcttcaggcattctatccagccccaggactctagaggcaaaagtagtctgatgagtaaccagaccctcggggtctcttcaaaaccactgaaaactcagcccgcctcaaaagccatgacggacaggcggctgttctccgagctagtagctggagagacacttcccaggacgacagatgagcaggagaaagcttctctagtgcagtgtgtgcaggtaaaaatgttcacggagaatcttctggaaggagtgatgtttccccagcccaccaacagcatgtctcggcaccctcaacatatatgaccccaaattagaggtatcgttctcctttagtgattgcaagaaaatggcagagaaatcaggtaacgcagcagcagtgttccgtgtgctttggtagaggctgatccacagttttactagtttactcactcatcctgcatctgcttttctgcagagaaagtttagtggtcggtaacggcccttgttccttagttccactctcaccttcagggctctgcattctgagcctagttctgatatcacatctggctgaggcacagccagttatggcattcattatcctggcagaggaaaacctggagtcttgggatagaggtggagacaaatgaccatctgaaagaaaactctagaaaatttgtatgccaaaatgctggtaattttagccttcggacaagttaaatagcagctcagtcagatgtgccatgtagacttctcttgttgtactgagccctgaacatgcctgaagtaggcagacttcatcactgagctcagtccagcccaccaattgttcttgtctccacagacattttgctgtcggctggaagagctgatctgctggttgtataatgtcacagatgttgctgacctcagtgctccacccaggaccagccttacaggtctcaagtcttctctgcagctttacagggtaatatgtagtcctggcttgtttatccacggggatgggagttaagtcacttttacaaaataaaatctcaagtaactggagtcttgtagctcagttgactattttatctgataaatcgaattaagatgtccttttcaaattctcagtgttgaacactataaaatgtactaagtgaggtaagaggataattacatacattggagccagaatgctttagaggagtttgcatgggattctggcagatccctcagggctagaatcctgggCTTTGATTTACAGACACTAATCAAGTATGACAATTAGTTAGGGTGAGCACCACAGCTATGCAGTGGCCACCACACCAGGCAGTGCAGACACAGAACGTGCCAGCCTCACCGCAAGTTCTTTTGGACTGTGTTGGTGTTTATGGGGTTGAGCTGAAACTACTGGGATCCAGGCACAGACAAGGTGATGTGAGACTTTGGGTTTTATTTCTGCC

This mutation is a 2580 bp deletion beginning at Chromosome 11 position 20,238,200 bp and ending after 20,240,779 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46234 CCT TAG ACC AAC TTC GTT TTC A Common A
46235 GGA AAG GGC ACT GTG TTG Wild type Reverse A
46236 CAC TGC ATA GCT GTG GTG CT Mutant Reverse A
46237 Fluorophore-1 CTT AGA AAC AAG AAG TCT GCT TGG TT Quencher-1 WT Probe
46238 Fluorophore-2 CCC CCC CTT TGA TTT ACA GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46234 0.40 uM
46235 0.40 uM
46236 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.