Protocol 35801: Probe Assay - Abcc8<em1(IMPC)J>Alternate1
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

>chr7:46178283-46178382 100bp GGCCTTCTGTAAACCAGTACC ATCTGCACCCCTTTATCCTC

Mut= 99 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CGTCTCAAAAATAAATAAATAAATAAATAAATAAATAAATAAAGAGCTGCTGCAGAAGGAAGCTGCTAGTTACTGACAGAGGTAAATGATGTGCATGCCCATATGTCTAGCAGAAACGAAACAGAATGGGTAGACAGTCACACAAGAGGTCAGTGTGGTGGTCCCTGCttaggaccggcctgcaggctcaggctttgtgcctggatatccagttcatatgctcatccctggggaaggaggatttgcatggtctccttatcaccatgctgggcctccaggtgggcgtcctgccttctgcgtctctccccacaggatggggcagccagagctccaaggtacacattcaccatagcacctggctccatttccctggccacaacctgcgctggatccttaccttcatgctgctctttgtcctggtgtgtgagatcgctgagggtatcctgtctgatgggtaagtgagctccaggggagtggggaagctgcaggagaccctccctgtgcctgctcatcggccttctgtaaaccagtacctggtgtgaactgcgccaggccttaggcacatatgagacgcAGGGCCTACCATCAGTCTGGAGGATAAAGGGGTGCAGATTTCTGTACCAGTTCCCAGTGAGACCATTGGAGAGCCTTGTCAGAGTGTCCCAGCTGCTGTGGGCAATACCCGGTCTCTCTGAAAGTGACTGAAGTGCGTGAGGGGACAGTAAATCCTGTCACTTGTTCTTACTGAAGGCCTCTCCTTGGTTCCCA

This mutation is a 406 bp deletion beginning at Chromosome 7 position 46,178,322 bp and ending after 46,178,727 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44885 GGC CTT CTG TAA ACC AGT ACC Wild type Forward A
44886 ATC TGC ACC CCT TTA TCC TC Common A
44887 AGC AGA AAC GAA ACA GAA TGG Mutant Forward A
44888 Fluorophore-1 AAC TGC GCC AGG CCT TA Quencher-1 WT Probe
47061 Fluorophore-2 AGG TCA GTG TGG TGG TCC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44885 0.40 uM
44886 0.40 uM
44887 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.