Protocol 31207: Probe Assay - Ankrd45<em1J> Probe 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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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=  105 bp

Wild Type = 142 bp

>chr1:163081235+163081376 142bp CTTCTGCAGCCCACTCTCA TGCAAGCTGCATACAACAGA

Sequence

Wt Sequence: TTTGATGCTAGGTATTTTTTTTTTTTTTTTTTGGAGTTAATGGAGCCAGAAGAAACTCTAGAATCGGAGAGTTCAGAAAAGTCACTATTTTCCTCACAGCAAGAGTATGAAGAGTCGCAAGAAGCAGAAGAAACAGGCGCTGAGAACCCCCTTCTGCAGCCCACTCTCACTGGGGACGTGGAAGGTTTGCAGAAGATTTTTGAGGACCCTGAACATCCTCACCATGAGCACGCTGTGCAGCTGCTTTTGGAAGAAGACATTGTTGGGAGAAATCTGTTGTATGCAGCTTGCATGGCTGGCAAAAGTGACGTAATTAAAGCCTTGGCAAAGTATGGGGTGAATCTGAATGAGGCAACTGCCAGAGG

 

Mutant Sequence:aaaagaaaaaggaaaaaagAAaacaccaccaccaccaaaaaacCCAAGTCCCTGCATCCCACAagactagtttagtgtagttccctctgctgaaagattaagtaagcaggggttgggatgtagctcagttggtacaacttgtac

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
31240 CTA CAT CCC AAC CCC TGC T Mutant Reverse A
31241 Fluorophore-1 TGT AGT TCC CTC TGC TGA AAG AT Quencher-1 MUT Probe
36385 GAA AAC ACC ACC ACC ACC A Mutant Forward A
36386 CTT CTG CAG CCC ACT CTC A Wild type Forward A
36387 TGC AAG CTG CAT ACA ACA GA Wild type Reverse A
36388 Fluorophore-2 CCC TGA ACA TCC TCA CCA TGA G Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
31240 0.40 uM
36385 0.40 uM
36386 0.40 uM
36387 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.