Protocol 32103: Probe Assay - Srek1<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

Mutant=  120 bp

Wild Type = 111 bp

>chr13:104549546-104549656 111bp GGCGTGTGTAAAGCACTTGT GACTGATCTCTCATCTCCACCA

Sequence

Wt Sequence (deleted region in lower case)

 

CACAGAAGCCACTGTGCCTAATAAGGGAGGTTTACCAGGTGCTCTTCACGTGTGACTCAGACATGGGACAGTATTGAGTAAAATGGCCATGTCATCACCTTCCCCTTCATCCTGCTAGGATATGCAGGATCTTAGCACAGTGTACTTAAGCATAGACGTGGTCCATCTTAGTCCTGATTACCAGCACTGTCCCTGTCTTGTAAGGCGTGTGTAAAGCACTTGTAAACTAGTAGTGCTCTATttagttgtggaaggcataagatagacttcagtttttcttgctttggtttcttggtggagatgagagatcagtcatcactttctagggttacttttggcgattctgtatttagctatttttatttccttatgtgctgttgagatgtcctcagattgataaacatgaatatcatactaacttttgtttcgtgctttataactattgtgtgtgtttgtgatatactaatatttttaatttagaataaatcactccaacaatgcaatagtaaaacctcctgagatgacacctcaggctgcagctaaggagttagaagaagtaatgaagcgagtacgggaggctcagtcatttatctcagcagccattgaaccaggtaaggccacagtgTTGTTGTACAGGACGTAGTCTAAAGACCATAGACTCTTAGAACTGCAAGGAATTGTAGGATTTCACTGCTCAGTTCCCTCATTGTACAGATCAGAAGACTGAAACTCAAGACATAGTAAGTGGCTTACTCAGAGCAACTCGGCACAAACCAGCTTTGCCCTAACACTGGGCCATGCATTTTAGCCACAGTCAGTGAGCGCTCTAGGAAAACAAGTCAGTGCTGAGGCTGCTCTGCTCAACAGCAGGGTGGGGTGTTTCTGCGAGGCTAGAAAGGAAGGA

384 bp deletion beginning at Chromosome 13 negative strand position 103,759,538 bp and ending after 103,759,155 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38341 GGC GTG TGT AAA GCA CTT GT Common A
38342 ATG AGG GAA CTG AGC AGT GA Mutant Reverse A
38343 GAC TGA TCT CTC ATC TCC ACC A Wild type Reverse A
38344 Fluorophore-1 CCA TAG ACT CTT AGA ACT GCA AGG A Quencher-1 MUT Probe
38345 Fluorophore-2 TGG AAG GCA TAA GAT AGA CTT CAG T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38341 0.40 uM
38342 0.40 uM
38343 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.