Protocol 35157: Probe Assay - Cwh43<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

>chr5:73411749+73411845 97bp TGTGCTAATTCTACCTTCTTCAGG ACAGGGAAAGGAAGGGTGAG

Mut= 95 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TTTGGCAGGGGGTGGAGAGAATGTTCTAAAATTAGCGGTGATAGTTGACAACTTTGAATGTACCCAAAACCTATTAAGTTGTATATTTTAACGTTGCGAACCTTATATGAGAATCCTACCTAAATGAATAAAGATTAAAGGTATGTATAGGTATGTGCTAATTCTACCTTCTTCAGGGCTAAGACcgtactagacacagttcttatcaaatgcagactgtcaggtgccagctcacccttcctttccctgtttgtaggcagcatagcttcctttgaggctccaaatgctaagcttcgactgatggtcctcgcccttggtgtgtcttcttccctgatagtgcaaactgtgacctggtggtcaggaagtggattgcaaaggtatgactcaggtgttttgtacctatagacaaacaggtgtgaccagtttccctggcgaagttctgacCATTGGTTTCAGGGCAGTTTTCCCATTCCTTTTCAAAAAATAAAGGTTCAAATACTTGCCACCAATGTCATTTGCTTTTGGTGCTAGGGACCGAACTCAGGTGTTGTGTGTATTTAGTTTCTCCCTGCTGTTTTGCTGTACTTCCAGCCCCAATAAAGTATAAACAT

This mutation is a 259 bp deletion beginning at Chromosome 5 position 73,411,781 bp and ending after 73,412,039 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45400 TGT GCT AAT TCT ACC TTC TTC AGG Common A
45401 ACA GGG AAA GGA AGG GTG AG Wild type Reverse A
45402 GGT GGC AAG TAT TTG AAC CTT Mutant Reverse A
45403 Fluorophore-1 ATC AAA TGC AGA CTG TCA GGT G Quencher-1 WT Probe
45404 Fluorophore-2 CAG GGC AGT TTT CCC ATT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45400 0.40 uM
45401 0.40 uM
45402 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.