Protocol 31794: Probe Assay - Cdk18<em1(IMPC)J> Alternate 1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  124 bp

Wild Type = 118 bp

>chr1:132121962-132122079 118bp GCAGTATCAACAACGGCAGA TCTACAGCAACCACCCTGCT

 

Sequence

Wt Sequence: AACCAACTCCACACTCAGACGAATGAGGGTGAGGGTCAGGCCACCTAGCTCTTTTCCACCCAGGCTCCCTGTGACCCTGATGACTAGCTGACATTGGgaagaaggggtgggggtgctctgggctgttcacatactctgggtgggaggtagggactgacacttgtcccactcagacatgccactcagtcttgtgggctgtcatcaggcgctttctcccccacagatggcacagatgagcctgagcagctgtctcctggcatgcagtatcaacaacggcagaaccagcgccgattctccatggaggtgagggtgcccctcccgggCTCCATGGGTCACACACGTCCACAGCAGTAAATTTAGCAGGGTGGTTGCTGTAGAGCCTGCCACTGGGGGGCTCAAACTGGTGCAGCTTATGGTGGGGGTTCAGTGAGAATATATGTCAAGTGCTAAAA

 

Deleted Region: gaagaaggggtgggggtgctctgggctgttcacatactctgggtgggaggtagggactgacacttgtcccactcagacatgccactcagtcttgtgggctgtcatcaggcgctttctcccccacagatggcacagatgagcctgagcagctgtctcctggcatgcagtatcaacaacggcagaaccagcgccgattctccatggaggtgagggtgcccctcccggg

226 bp deletion beginning at Chromosome 1 negative strand position 132,122,242 bp and ending after 132,122,017 bp (GRCm38/mm10).  In addition there is a 17 bp intronic deletion 20 bp after the 226 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37058 AGG CCA CCT AGC TCT TTT CC Mutant Forward A
37060 GCA GTA TCA ACA ACG GCA GA Wild type Forward A
37062 Fluorophore-1 CTC CCT GTG ACC CTG ATG A Quencher-1 MUT Probe
37063 Fluorophore-2 CGA TTC TCC ATG GAG GTG A Quencher-2 WT Probe
37672 GTG GCA GGC TCT ACA GCA AC Common A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37058 0.40 uM
37060 0.40 uM
37672 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.