Protocol 34341: Probe Assay - Anapc10<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

>chr8:79719381+79719489 109bp ACCACCCAAAACATTTGTCA GGCTGAGTTTGTTGTTTACTGA

Mut= 89 bp

Wt= 109 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CCGGCATAGGATGGATCATTCTTGTGATTAGTTTATAAAAAGGATAAAAACTTGGACTTGTGCAAATAGAGACCTTATATAACTCCAAAGATGCTTTGAAAAAACTAACCACCCAAAACATTTGTCATAGTATAAACTTTAGTGTGTGTAAGGGattgagcccaggacctcccatttttaaaataaacagtctatcagtaaacaacaaactcagcccattaggggatttttagagttgaaatctggatgaaagaatagaattctgcttaaatttcatattctaactgtcttctatgtcaacatttctagacaaatatgtatggtaattttatgttttaaatatgtaactttttcatggaaatttgtcattttcactgtttctagggtttggagtggatcaattacgggatgacaatctagaaacttactggcaatctgatggctctcagcctcacttagtgaacatccaattcaggtaatccatttcttacatttttttatatagtgtaggttgtaagtggacaaggaaatctattttattttcataaaagttaagaaaacatggaaagtaaaatgaaaatacttaattgctttcagactctattaggtcttagagatttttttttttttttgccatccagATTGCAGGTAGCTTCAATGTATTCGGCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCAGTAAGTCAACAGGTTCTCCAGTGCAGGAGTGAGGATTAAAAAGACAGATAACATTGCAGCGTGACCCCAGGCAATTCTGAGACTGAAGGCCAATTTAATATTTCCAAT

This mutation is a 487 bp deletion beginning at Chromosome 8 position 79,719,428 bp and ending after 79,719,914 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43358 ACC ACC CAA AAC ATT TGT CA Common A
43359 GGC TGA GTT TGT TGT TTA CTG A Wild type Reverse A
43360 GAG AGA GAG AGA GAG GCC GAA TAC Mutant Reverse A
43361 Fluorophore-1 ATT GAG CCC AGG ACC TCC Quencher-1 WT Probe
43362 Fluorophore-2 TGT AAG GGA TTG CAG GTA GCT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43358 0.40 uM
43359 0.40 uM
43360 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.