Protocol 34002: Probe Assay - Kctd19<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

>chr8:105396607-105396694 88bp AAAAATGCTTCAAACTCAGAAGA TAAACGGAATCAATCCCAAAATG

Mut= 105 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AAACAAAAAAAAAAAAAAAACAAAAACAAAAACAAACAAAAAAAAAAAAACAAAAACAAAAAAAACCAAAAAACTCCATAGATTTTTTTTCCAAGGAAAATATCCTGTTATTTGATATAAAATTAGAAGAAAAAAAATGCTTCAAACTCAGAAGAAAAAAAATCattgagaattttaattattaccttgccattttacattttgggattgattccgtttattttaaaataccgtcccaaagcaagtcacatgctattttttctgagtattcagtgtcaaaacaaaacaaaacaaagttggttatgaggttttgatgttgaaggcagcactcttctctgtgggacagaccctagataacctgaaggaagggagacaccacctccgtgtgcggcctgccgacattcctgtggctgagagagcatccctgaactactggcggacatggaagtgtatcagcaagccctcggattttccaataaaaagccccgccttcacaggtaccgtcacgggcaggcccacaccctgaagtcaccagcttcgactctcctcgcccgaacagacgttaacctccatctgaactacttctgggtgctgcctctcttttcctcttctcttttgacccagtactgtcctctgcatggctgcttgcttactgggactcagcaccctgtgccattccgtcctgctgtatctaggcttcctttaggtgatatgatgtcatccAGTCTCTCCTGGCTCCATCATTATTTCAACTTACCTCAGACCACTACACAAAAATGCTCAGTGGTCTCTGGCTTCTGAGGCTCTTGCAGCATAGGGACATAAGTCCCTATGCTGGGACATCAGGAG

This mutation is a 559 bp deletion beginning at Chromosome 8 position 105,396,104 bp and ending after 105,396,662 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42632 AAA AAT GCT TCA AAC TCA GAA GA Common A
42633 TAA ACG GAA TCA ATC CCA AAA TG Wild type Reverse A
42634 AGC CAG AGA CCA CTG AGC AT Mutant Reverse A
42635 Fluorophore-1 ATT GAG AAT TTT AAT TAT TAC CTT GCC A Quencher-1 WT Probe
42636 Fluorophore-2 ATC AGT CTC TCC TGG CTC CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42632 0.40 uM
42633 0.40 uM
42634 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.