Protocol 35849: Probe Assay - Clcf1<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

>chr19:4219956+4220072 117bp TGATAGACACAAATGCCAGGA GTGAGGGAGAGAGACCAGATG

Mut= 115 bp

Wt= 117 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTCATGGGACTGGAAGGCTTGGAGAAGAGACTGGGACACAACTGAAGCTTAGGCATATTGACACAAAATCCCTAAAGCCTGGCCTGCCCCTTTGAATAGACACATGCACACATATTTGGCAGATACATGCCAAAGACTTTCAGCTAATAGCAGTCAAGTATCTACTGATAGACACAAATGCCAGGATTTGTCTTTGCATACATACCTCcacatacgctcttggaccagaagtctttcccctcctccaccccctccatttcccatctggtctctctccctcaccccccttcctagcccaaggtggggacagacacctgaggggctgccagctgtttccctatttgggcctgggcccaccctcatacttcttgcccatcctattcacaggggactcgtgggggatgttagcttgcctatgcacggtgctgtggcacctccctgcagtgccagctcttaatcgcacaggagatccaggccctggcccctccatccagaaaacctatgacctcacccgctacctggagcatcaactccgcagcttagctgggacctacgtgagtatcccctttggcaaagaggaggcagaggctttggggaagagagagcatggggcaggggtcctagtgaatgATGGGGTGATGAGAGGCCCTTTGGGTCCACATAGCTTCTCCATCTGGCCTATCTTCTGCCCTTCCCTCTCAGGTGCCCCCCCCCAATCCTGGCCCCAGGAGTAGGCATGTGGGCAGGCCTCGAACCCGCCTTGGCCCATTGCCCCATTGGCTGCCAGCCCAGCTGCCTGCCTC

This mutation is a 422 bp deletion beginning at Chromosome 19 position 4,219,999 bp and ending after 4,220,420 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47153 TGA TAG ACA CAA ATG CCA GGA Common A
47154 GTG AGG GAG AGA GAC CAG ATG Wild type Reverse A
47155 CTG AGA GGG AAG GGC AGA AG Mutant Reverse A
47156 Fluorophore-1 ACG CTC TTG GAC CAG AAG TCT Quencher-1 WT Probe
47157 Fluorophore-2 CAT ACC TCA TGG GGT GAT GAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47153 0.40 uM
47154 0.40 uM
47155 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.