Protocol 34398: Probe Assay - Ccnj<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

>chr19:40836728+40836843 116bp GGAGGGAGAGTGTATTTGCAG CATATTGTGTAGTGAGAGCTTAAAGAC

Mut= 107 bp

Wt= 116 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GGAGCCTACTCACCAGTCCGCTCCCTTTCAACCAAAACTTTGACCTCCTTGGAGGGAGAGTGTATTTGCAGTGTGGTTTTTCTTAAAGCCATACACCTTCCACTCAActttattgctgcgctcctttcgagcaaagtgtgtctttaagctctcactacacaatatgttaggaaaacaacgttaagtcttgatactctgaagatacgtctatatattaaatattcctcctcttgatctaggagctgaagctgccttcttacaagggccaatctcctcagctgaacctcaggcggtattttgctgatttgattgccattgtgagcaatcgcttcaccctctgcccccctgcccgccatctcgccgtctacctgctggacctgttcatggaccgctatgacatctccattcagcagctacacttagttgcgctttcctgtctgcttctagcaagtgagtatgaactggagtgacccgactcaaggccgacagcgctgacaggatgaaaccggagaactcaccgcattattgtcaaatcccagtcacttcccatttattgcttttaggaaatttatattacaaatttattttatggttgttagttagatattaaaaaatagttcaggcatttttttttaaaacaacaaatgcctaaaccctacctcatcaaGATTGATACAAATACAGATTCTAGGCACCACATACAGTACAGGTTAATTGTCAACTAAACGTGATTTTGAATCTCCTAGGAGACACACTCTGCACATGGGAATTTCCAGAGAGGTTTAACTAAGGAGAAACCTACTTTGAGTGC

This mutation is a 560 bp deletion beginning at Chromosome 19 position 40,836,785 bp and ending after 40,837,344 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43501 GGA GGG AGA GTG TAT TTG CAG Common A
43502 CAT ATT GTG TAG TGA GAG CTT AAA GAC Wild type Reverse A
43503 CAA TTA ACC TGT ACT GTA TGT GGT G Mutant Reverse A
43504 Fluorophore-1 CGC TCC TTT CGA GCA AAG T Quencher-1 WT Probe
43505 Fluorophore-2 CCT TCC ACT CAA GAT TGA TAC AAA TAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43501 0.40 uM
43502 0.40 uM
43503 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.