Protocol 36410: Probe Assay - Scml4<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

>chr10:42924204+42924348 145bp CTAGGTGGCAGTGATCCTAGA AGATGTCAGCCTGCCTTTGC

Mut= 135 bp

Wt= 145 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTCTTCACCAGTTCCATTAGTGTAAACACCATCTTCTCACGGTGACGTTTCAGGAAGACCCTCACAGTGGTGGCTACTCCTTCCGATCCCCATCTGCATTGTCTGATGGTTTGCTACAGGAAGTCACTGCTGAACAGCTAGTCTGAAACCTGGAGAGAGCTCATGCTGTGAGCTCATCAGAGAATGGGAACAGCACCATGGCCTATCTCCTGGTGGATACAGGGAGAGCCTACAGCTGGGATTCTACCTAGCACAGAGCTGTGGGCAGAGCACTGCCCTGCAGTCTGTGTTCTAATCTTGGGAGCTTgtgtgtcttaactgttcatgcctctctgtgttcctacacacgttgcctggtgctgtcatttcccgggcagtgtcaatgtaagggtacagagctctcctctgaggataatccaaatacaaattttgaacatgaacttgaatctgaagctctgttcccaaagctgaggagtaggaaggtgcaggagtgtgaaggtgtagagagagttagcaggcagccaggatggtcagggtggtatagagagcatttttagccatttgctgattgaactgactctcctgcagatcaagtctccagtgctcatgactcccttggccctgtcacctccacggagtaccccggaacccgatctcagctccatccctcaggatgcagccaccattcccagcctggtggtcccagaggctctcacaggtacgtctgccaaaggacttctaggtggcagtgatcctagagtaacccggcgtcctttcatctaatggttactttatgaatctaagagtttctctgaggtacttatttcactggGTGTGATGACACATGCCTTTAAACCCAGGAGGCAAAGGCAGGCTGACATCTGAGTTTAGGGCCAGCCTGGTCTACAGAATGAGTTCCAGGATAGTCAGGGCTACAGAGAGAAACCCTGTTTCTGAAAAGAAAATATAAAGAGAGAAAGGAAGAAAAGAAAAGAAAAGAAAAGAAAAGAGAAGAAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGGAAAGAAAGGAAAAGGAAAGAAAGGAAAAGGAAAAGGGAAAAGAAAAAGAAAAAGAAGAACAAAAATTTCCTGAGATAGCTCAAAGAAAGAT

This mutation is a 525 bp deletion beginning at Chromosome 10 position 42,923,773 bp and ending after 42,924,297 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48694 CTA GGT GGC AGT GAT CCT AGA Wild type Forward A
48695 AGA TGT CAG CCT GCC TTT GC Common A
48696 GAG AGC CTA CAG CTG GGA TTC Mutant Forward A
48697 Fluorophore-1 CCG GCG TCC TTT CAT CTA A Quencher-1 WT Probe
48698 Fluorophore-2 CTT GGG AGC TTG TGT GAT GAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48694 0.40 uM
48695 0.40 uM
48696 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.