Protocol 34885: Probe Assay - Dclk2<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

>chr3:86906366-86906452 87bp TTCTGGGCTTAGGGGTTTTC CCTTTTCCTTTAACCTTCTCCA

Mut= 90 bp

Wt= 87 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGAAGTCCTTTATAATTTAGTTACTTGCTCTATTTAATTCTGCAGCCAAGGATACTGAGACTAGAAGTGTGACATAATGTCTTATCAAAAGTGACACATTTGGGTAAAATAAGCCCAGATCTCACTCTTAGTTTCTGGGCTTAGGGGTTTTCACATCTCAGAAAAGCAGTctacgtaattgagaagggagggaggtttggagaaggttaaaggaaaaggttgtaatgtcccttttcctgtgaccccccaaacttagggcttggcttgcaggtctctagaagctgttgtttacatataatggattcaggtctactatggccagtgctcagagatctgatattatgctaattattatttttgttttttaggtgaaagttacgtgtgtgcctccaacgaaccatttcgtaaagttgattacaccaaaaacgttaatccgaactggtctgtgaacatcaagggcggaaccacccgcaccctggccgtggcctctgcgaagagtgaggtgaaagaaagcaaagacttcattaaacccaagttagtgactgtgattcgaagcggagtgaagcctagaaaggccgtgcggatccttctgaataaaaagactgcccattcctttgagcaggtcttaacagatatcaccgaagccattaaactagactcaggcgtggtcaagaggctgtgcaccctggatggaaagcaggtaagtgcttctgcctgcagctcgattgttgattatccttgtgggtatttgaagagatgcctggaacttgctgtgtagtccaggctgaccttgaacttgggatcttcctggtcctgccaagtgctaggattagggtgtgcaccatgcccaactattgcagttagacattccacagaatgtcctggtctcctggaagtaatcctagtGGACTTTTTTTATGAAGGATTGTGTGTGTGTGTGTGTGCGTGAATTAGCCTTTAGCATAAAAAATATAAAGGGCTTCCGTTTCTCCAATTATCTAAGAGCCCATTATATGCCTCTTCAGTTTGTAAAAGGAAAAGATTGAATTCCTGGGGAATAAGTTGCAGTACTGTCCTATGCTAAG

This mutation is a 736 bp deletion beginning at Chromosome 3 position 86,905,679 bp and ending after 86,906,414 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44702 TTC TGG GCT TAG GGG TTT TC Common A
44703 CCT TTT CCT TTA ACC TTC TCC A Wild type Reverse A
44704 AAG GCT AAT TCA CGC ACA CA Mutant Reverse A
44705 Fluorophore-1 ACG TAA TTG AGA AGG GAG GGA G Quencher-1 WT Probe
44706 Fluorophore-2 AGC AGT GGA CTT TTT TTA TGA AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44702 0.40 uM
44703 0.40 uM
44704 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.