Protocol 32096: Probe Assay - Sccpdh<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=  130 bp

Wild Type = 133 bp

>chr1:181600444+181600576 133bp AATTCTCATGGTGCCGAGTG TGCTGAGTAGATGCCTGTTCTG

Sequence

Wt Sequence (deleted region in lower case)

AATGAAATCACGGGTCTGGCGAGGTGTCCCAGCAGTTAGAACATGTACTGCTCTTGCTGAGGATCCCAGCAGCTGCATTGGGTGACTGACAGCCCCATGGAACTCCAGCTTGAGGGAATATCTGCTACCTCTGGTCTCCTTGGGCACCTGCACTGACGTGCGTATGTGCACACACACAATTTAGTAAAATAATTCTTTAAAACTACTAAAAAGTGCATTGGCCTCCAAGTCCTCTTTAATTCTCATGGTGCCGAGTGCTGACGGCCTCGGCCTCctctctataattgggtcagatatagttgtttaggagctgtttttgtaatagagacatcttgatggaaaggaagtcagaacaggcatctactcagcatattaacagattataaaggtttcatggttgaaagtcttcagtgtaaggttgccatcactgaccactataggcaggtgatctaggaggtctcacgttgacttggacttgaatgacgtgattctgttttttaacgttaggaagaccatcactatcatctgaagttggagtcataatctgtgatatcagtaatccagcctcacttgatgaaatggctaaacaggcaaagcttgtcctcaactgcgtaggaccggtaagtagtcggcctttctttgtatcagaacaaacagtccattcattcctaacaaaatctggtattttacagtccagagggacacacgggtggagggcagatggcagattgaaggccttccctccacagggaagtctttccattgatatgatagtcttccaaagtagtgacaggtctttgagaccctattcagagaatgtgctgattggcaccccttaccctgtgtccccCTCTCTAATGTAAAAATAAAATAAAAGTTTGATGAGCTAGAAAGATGGTTCAGTGGGTAAAGGCATTTGCCCTGCAACCCGACAAGTTTTGTTCAGTCTCTCAGACCCATACAGTGGAAGGGGAGAACTGGTCCCCAAAAGGTGTCCTGTGATCTGTACCCTTGCACCTTAGCATGGGTACATGCATGCATGTGTGTGTGTACACTCCCATACACAGTAAATACATGTCATTTGACATTTTTAAACGTTTGCATTTACAGTCCCAGCTTCTTTGCTTTACTCTTGTATCATTATGTAGAT

576 bp deletion beginning at Chromosome 1 positive strand position 179,670,350 bp and ending after 179,670,925 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38336 AAT TCT CAT GGT GCC GAG TG Common A
38337 AAC AAA ACT TGT CGG GTT GC Mutant Reverse A
38338 TGC TGA GTA GAT GCC TGT TCT G Wild type Reverse A
38339 Fluorophore-1 TGG TTC AGT GGG TAA AGG CA Quencher-1 MUT Probe
38340 Fluorophore-2 AGA GAC ATC TTG ATG GAA AGG AAG T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38336 0.40 uM
38337 0.40 uM
38338 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.