Protocol 34435: Probe Assay - Akap8l<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

>chr17:32335340-32335455 116bp CCTTCCTTATGGGACTGGA TGCTAGACTGGAGGCCACAC

Mut= 114 bp

Wt= 116 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGGCCAGTGCATGCCTGGTGCCTCCCGGCTGCCCTCCCTCTTCTCCCAGAACATCATACCCGAGTATGGCATGTTCCAGGGCATGCGTGGTGGGGGTGCCTTCTCTGGTGGCTCCCGATTTGGCTTTGGATTTGGCAatggcatgaagcagatgaggcgtacctggaagacctggaccacagccgacttccgggtgaataagctgacctacccctcctgatccatgctggaatatggctacaatctctgagttcactcctcctttcacttccagaccaaaaagaagaagagaaagcagggtggcagtcctgatgagccagacagcaaagctaccagaacagactgctcagacaacagtgactcagacaatggtaagttggggaatgtggtgtcctgctccctgtccccgtggggcccctggattgaacttgactgtcctctccttgcagatgaaggcactgagggggaagctgcagagggtactgaaagtgctgaggctatggagaaaggctcccgagcagtaagtaaccctagacctcaggctcttgctctctgcaggtggggcataggactccagagctggcttaaagacacacagccttgctacaagggtcacaggcaatgtggtcttacctgtatgcatggctagtgagtggccagcacaaggaggctgggtatctagggaaaagtacagaccacttcactaacagtgtgtttccttgggagaaatggggcgatgcaggcctctgtgcagcaggtagtgagtgggaaggtctgacgctcccttaatcctttagagcaggtataagtagctgtgccctttttaaaattgaattctaaaaattgtatattaggaaggagaagacgaggagggaaaagaggatgggagagaaggcaaagaagattcggagaaaggtaagtcttccatggttacctaggtactgggaaagctcttccaggtgcttgtagtgggaggaaaccagctgagggcagggttgtagcggaaagatggctggctgggaccttccttatgggactggatatatgttagcaaggcctcttcttactctgtgccctcccagcctgcctccgtttttcctaGGCAGAGGTTATGGATGGTGTGGCCTCCAGTCTAGCACTTTGGTTTTGCCTTCAGGTGGGGGCAGCAAGCAAAAGTGTGGGGAGCCCAGGCACCCCCCCCCTCAGTTCCTCAGTAATAGACAGTTCCAAGTCCTC

This mutation is a 964 bp deletion beginning at Chromosome 17 position 32,335,377 bp and ending after 32,336,340 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43614 CCT TCC TTA TGG GAC TGG A Wild type Forward A
43615 TGC TAG ACT GGA GGC CAC AC Common A
43616 CGA GTA TGG CAT GTT CCA G Mutant Forward A
43617 Fluorophore-1 CTC TGT GCC CTC CCA GC Quencher-1 WT Probe
43618 Fluorophore-2 CTT TGG ATT TGG CAG GCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43614 0.40 uM
43615 0.40 uM
43616 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.