Protocol 31806: Probe Assay - Cntnap3<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

Mutant=  150 bp

Wild Type = 158 bp

>chr13:64799187-64799344 158bp GCAAAACTATTTGAAAGAAGACAGC TGTCTGCATTTGTGTTTCCTG

Sequence

Sequence (deleted region in lowercase, indels in parentheses)

GACAAAATTGGTGCATACGTGCTATGGGATGCCACAGTACCAGGAGACTAAGTTTCCGTGAAACTGTTCACCTCGGGACTGTGTCCAGGTTCCTAAATCTGTCAAGCAAGTCATTACTGGAGTGGATGTAGCAAATAGTAGATAATTGAGAAAGCAAAACAAAATTGCTATATCTATGTTAAACATTAGCAAAACTATTTGAAAGAAGACAGCCTTTT(ggata)TAGGCAGAGTGAACACATGCCACCATTGAtataagctatgataatacaaaaatgatcttgaagatacgttgacccttcctctttgttttttacagggatttccaggaaacacaaatgcagacagtgtggtacaatatagtctccagccttccttccacaccaggttcctgcgcttcctccccttaacctggaaccctaaaggcaggattgggatgcggatagaagtatatgggtgttcatacagtaagtgatatttattttccatgtaaaataaacatctgcatgagatgctgcaaaagccagcgtcAGTTAAAAATTACTTGCTCTAGACATTTTTAGTAATTGTATAATTAAAACCGA(gt)GTTATGAGACTTCTACTAACTCCTTATGACAATATAGGCTTTAATTATTTTTTTCATAGTTAAATTTAGAGATTGCTTAGTGAGAAAAATGTTTGATAGGAAATAAGGGTGTAATTTTGGAGCCCTAGTATCACATAAAAGGGCAGGTGTACAGTGAATGTCTACGAACCCAGTGCTGGAGACAGAGAACAGAGACAGGTAAATCT

278 bp deletion beginning at Chromosome 13 negative strand position 64,799,280 bp and ending after 64,799,003 bp (GRCm38/mm10). There are 2 additional small deletions, a 5 bp (GGATA) deletion 29 bp before the exon deletion and a 2 bp deletion (GT) 53 bp after he 278 bp deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37711 GCA AAA CTA TTT GAA AGA AGA CAG C Common A
37712 TGT CAT AAG GAG TTA GTA GAA GTC TCA Mutant Reverse A
37713 TGT CTG CAT TTG TGT TTC CTG Wild type Reverse A
37714 Fluorophore-1 TGC CAC CAT TGA AGT TAA AAA TTA C Quencher-1 MUT Probe
37715 Fluorophore-2 TGA TCT TGA AGA TAC GTT GAC CC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37711 0.40 uM
37712 0.40 uM
37713 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.