Protocol 31906: Probe Assay - Csnk1g1<em1(IMPC)J> Probe 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=  141 bp

Wild Type = 163 bp

>chr9:65999736+65999898 163bp AATTTGGCCACAAAGCCTCT GGAATGGTTCTGCTTCAAGG

Sequence

Wt Sequence (deleted region in lower case)

ACTCCTAGATGAGAAAAAGAAACCCCTACAGAGATTTTGTGTGAAAAGAAAACCAGTTGTGCTGGCCTAGGGCCAGGCTGCACCTCTTTGGCCTTGCCTCTCAATCCGATTCTCCAGGGGGCAGCAGTTACCAACAACTCTTTTTGGCTTTAGCAGCGAGCCCAGGGAGTGCTAATGAAACTAGGCGGATGTACCAAGGTTTGTTAGCTTGCATTTGAGCTCGGTGAGGCTCCCTTTGAACCTTTTATGATGCTGCACTTTCAGGCATCCTTTGGGAGAATAGTTTACATCCTGAAGCAATAATGGATCAGAGACCATTTTCTTTTATACAGCAGTGGAAACGCCTTTGGCACTCTGGTATTAAATAACTGCTTATTTGGACACATTGTGCAATGGCTTTGCCTTTTCCGCccagtaatagttcccacagaagctattatactcagtttaacttttccaGGAgggactctgaaaggaaattgaaatcttagagattatactttatattacattcattgctaatgtcttatgttttagtttttgtgtctctcactctgtaccccatttgcccctctactttcaggtgaaggcctcccacaggtttattactttggaccatgtgggaagtacaatgccatggtgctggaactccttggccctagcttggaagacttgtttgacctctgtgaccgaacgtttactttgaagacggtgttaatgatagccatccagttggtaagttgatgtgtggttccagaagataaatggatcccttgcccacctccttgagggccctttaatgatgtattagctgcagtgagatctggtgacttcgtttttacaggcttaatggcattctagaaggatctagcagacttctgaaaggaactattgcagctgctgctttagctggttcctgtgtataatttggccacaaagcctcttggccagtatgtaatatgccaaacagctaaggtaggttcttagccacacaacttaaagggttacagagggttaactgagggAAGTCAATGCATCTGCAGTTTGCTCTATTTTAAGAGTTTGCACCTTGAAGCAGAACCATTCCTGTAACTGAAGTGGGGGAATATAATTTACATTAGGGAGAAAGAGGACGATTAGGTAGAGCTGTGTGAATTCAAGTAATTGCTTCAGTGTCTTATTTATAGGTCTTAGGCACACAGTTGTAAGCTTTTATCATGCCTTCTTTCTCTCACTGTTGAAAACCAGAAAAGACTGAAGGTACCAACCTTTATTCAAAGTCACGTAACTGAGATGTTACAGATAATATACCATATATAACATGTAGCATATTATATACTTGTGGGCTGACCAAGAAGCTTTACAGCTAAATGAAATACTAGTTCTACTT

611 bp deletion beginning at Chromosome 9 positive strand position 65,999,223 bp for 48 bp where there is an endogenous 3 bp retention (GGA) followed by 563 bp deletion ending after 65,999,836 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36707 CAG TGG AAA CGC CTT TGG Mutant Forward A
36708 GGA ATG GTT CTG CTT CAA GG Common A
36709 AAT TTG GCC ACA AAG CCT CT Wild type Forward A
36711 Fluorophore-1 AGG TAG GTT CTT AGC CAC ACA ACT Quencher-1 WT Probe
37947 Fluorophore-2 TGT GCA ATG GCT TTG CCT TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36707 0.40 uM
36708 0.40 uM
36709 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.