Protocol 30190: Probe Assay - Alpk2<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 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

 

>chr18:65372829-65372988 160bp CATTAGCATATCCCATTGTGC TTGGGCTGACCTGAAGATT

Mut= 161 bp

Wt= 160 bp

Sequence

Wt Sequence (deleted region is in lower case and insertion is indicated with ^^)

ATCATATATTTTATGTGTGTGTGTGCATACATGTATACCGCAGAATATATTTGGAGGTCAGAGGGATAAACACTTTAAAAATTTTAAAGTATTTTCAACAACATGGGAGTTAGTTTTCTCCTTTAATCATGCAGAGTCCAGGAACCAAACTCAGGCCACTAGACTTCAATAGCAAGTGCATTTATCTGTAGAGCCACCCTAATTTTGGAGGATTATTTTATTTTGGATAATCTAGAAAATAGTTTCTATATTAGAGCATTTTTTATTCTTATTTCATTAGCATATCCCATTGTGCTCA^ATTGTAAAAGGGTCAGTTGAAGTTATTA^tcaataggccttcttattaattcctatttataatttattaattcctatttataacttcaactgacccttttacaatttttttttgaaaaatatttaatatcctttaatcttttttttaatcttcaggtcagcccaagccagaggtgacttggtataagaatggtcaagccatagacttgggaggcactgtttccagttatgagttctttgagaaccagtatattcatttgttgcatctctcgtggtaagtggcttggcctctgtgaactactcactaaaattctgtcttccatggtgtgctcatgtttgcctcagaagtcctggactttgatgttactgtcaggcaacagcctaaccagccagtactgactaaagtgttccctctgtatgaatggattacctgaatatttaatgaaccgtgaggctgaaaatatttttaagaattgtattgggattagggatggttatcttcactccctagacagtatagcacACTAGTAATTTACCTATAATTTATTTCATATTAACATGGGGGTGATTTGTACAAACAGTATATCATATTATATAGGAAAGAGACTTAAGTATCCTCAGGGCTGATTATCTGGGAACTGAGATCTCCATAGGACAATGAAGAGTGATCATATTCATTAAGCTAAGTAACTATTTGGCCTTCTCTAGTTTACCATTGATAGACTTAAACTTTATCTAGAATTCTAGGGTT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
34075 CAT TAG CAT ATC CCA TTG TGC Common A
34076 TTG GGC TGA CCT GAA GAT T Wild type Reverse A
34077 Fluorophore-1 CAT CAA TAG GCC TTC TTA TTA ATT CC Quencher-1 WT Probe
34078 Fluorophore-2 TTT CAT ATT AAC ATG GGG GTG A Quencher-2 MUT Probe
34079 GAT AAT CAG CCC TGA GGA TAC TT Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
34075 0.40 uM
34076 0.40 uM
34079 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.