Protocol 44095: Probe Assay - Ankdd1b<em1(IMPC)J>-Alternate1
Version 2.0

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

>chr13:96420476-96420613 138bp CCAGAGGTCACTGCAATAGG CCCACTCAATAGGCCAAAGT

Mut= 126 bp

Wt= 138 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

GTTAAGGATGCATACTAGGGGCATTGATGAGAAAATCAAATACATCAAAGAGAGAGGCTAGTGTTGAGTGTAAGGCCATTAAAGCCATTGGCTGTGATGTCTCACTCGCCATCATGTCCCCTCAGCTCTTTTTGAAAGTGGGTGATATCAGTCGGCAACACAGACACAACACTGCAAACTCACTCACGTTTGCTGTTTCAGACACAGTACACACACTTCTTGTTCCAGACATTTCCATCcaacttggatgacaaaagatgttctaggacagactgaaaggagtcagctgctgccttaccaatcctttatgacgtaaggtgacaagtgagcattttagagtcaatagctactgctaattttcaacttaggagcaccgtgagagcatccaggactcagcagtgagctctacactgacgttcaagcaggaccatagtctggagaccaggcaaatccgcacactgctctggaacctggcctatcggcagctgaaaaagaaagactggcagaggctggcccgcttgtggagttttacagaggaccagatcagagccattgaggaacagtggtcaggtgagtggcggccagatgacatggacaggatgaagccagttaaagttaggagtaatggtctttccctcctcaagagtcagtgcctggtgcacacaattggcagaagccagaggtcactgcaataggggtagttagcatggctcagtctgcaaaggtggtttggaatttagcagagagctacactcctgagaatGTTCGGTTAGCTGTGCTTGCAAAGGTTGACTACTTTGGCCTATTGAGTGGGGACCAAAGAAGCAAGGACACAATCCCTGGAATGCCTCTGGAATGCCAGTTTGGCTTTCACTAAAGTTTGCAGGTTGTTCTGGGATGAAAGGTCTTCCTCCGCCACAGAAAGCTTTAGTACCGAAGGTT

 

This mutation is a 524 bp deletion beginning at Chromosome 13 position 96420527 bp and ending after 96421050 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64405 CCA GAG GTC ACT GCA ATA GG Wild type Forward A
64406 CCC ACT CAA TAG GCC AAA GT Common A
64407 CAC AAC ACT GCA AAC TCA CTC A Mutant Forward A
64408 Fluorophore-1 CTC AGT CTG CAA AGG TGG TTT G Quencher-1 WT Probe
64763 Fluorophore-2 ACA TTT CCA TCG TTC GGT TAG CTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64405 0.40 uM
64406 0.40 uM
64407 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.