Protocol 31524: Probe Assay - Scrn2<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 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=  127 bp

Wild Type = 142 bp

>chr11:96892447+96892588 142bp CTTGCCCTTGAGGCCTTTCT CAGCCAAGGCTACACAGAGA

Sequence

Wt Sequence: ggagcagatggagtggaacgagtacggagttccagtccaccaacctggacccttgagtggagttagcttctgctaactcggtttacttatgtgtgacctgagaataagacctttctcagggccatttaaagtgGCTGACAAAGGGTCCGGGCAGTGTAAGTTCTGGGTCTTTTTTTTTTTTTCACTGAATTCTTCCTCTCTCTTCTTTACCTCCAGTGTACCTATATCGAGGTGGAACAGGTGGGGAAGACACACGCTGTGATTCTGAGCCGCCCCTCTTGGCTGTGGGGAGCTGAGATGGGGGCCAACGAGCTTGGTGTCTGCATTGGAAACGAAGCAGTGTGGACCAAAGAGCCTGTGGGGCAGGGGGAAGCCCTGCTGGGTATGGACCTGCTCAGGTAGAGAGCTGCTCTTCAGTGCCCCACTTCCTCCCACCTAGCCTGAGTCTATTGGGCTTGTCTCTGTTGGCCTTGCCCTTGAGGCCTTTCTAAGGCGGCTGCAGTCTTATATCTTCCTAGTCTGATAAAGGACCTCCCTTGgcctagagtgctttaatttttttttcagtactttttgttttgagacagggtttctctgtgtagccttggctgtccaggaatttgctctgtagaccaggatggccttgaattcacagagatcctcctgcctgtgtgccaccacacatagttcagtacaacttttgtttgtttgttttttggt

 

Mutant Sequence: GCTGACAAAGGGTCCGGGCAGTGTAAGTTCTGGGTCTTTTTTTTTTTTTCACTGAATTCTTCCTCTCTCTTCTTTACCTCCAGTGTACCTATATCGAGGTGGAACAGGTGGGGAAGACACACGCTGTGATTCTGAGCCGCCCCTCTTGGCTGTGGGGAGCTGAGATGGGGGCCAACGAGCTTGGTGTCTGCATTGGAAACGAAGCAGTGTGGACCAAAGAGCCTGTGGGGCAGGGGGAAGCCCTGCTGGGTATGGACCTGCTCAGGTAGAGAGCTGCTCTTCAGTGCCCCACTTCCTCCCACCTAGCCTGAGTCTATTGGGCTTGTCTCTGTTGGCCTTGCCCTTGAGGCCTTTCTAAGGCGGCTGCAGTCTTATATCTTCCTAGTCTGATAAAGGACCTCCCTTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37064 CTT GCC CTT GAG GCC TTT CT Wild type Forward A
37065 CAG CCA AGG CTA CAC AGA GA Common A
37066 CGG TTT ACT TAT GTG TGA CCT GA Mutant Forward A
37067 Fluorophore-1 CTT CCT AGT CTG ATA AAG GAC CTC CC Quencher-1 WT Probe
37069 Fluorophore-2 ACC TTT CTC AGG GCC ATT TAA AGT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37064 0.40 uM
37065 0.40 uM
37066 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.