Protocol 34607: Probe Assay - Kansl3<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

>chr1:36357723-36357823 101bp TTTCCCTCTATAGCCTTGTAGTTC CAGAAGGTACCCTTGGTCCTC

Mut= 100 bp

Wt= 101 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGTGTTGTAATCCCAGCACTATGGAGGCCGAGGCAGGAGAAGAGTTTAGAGTTTGAGTACAGTTTGAAGTGAGACCGTACTGGAAATAAATAGAACTGAAACTTGTCTTTTGTTACAAATGTGATTATTTTAGCCACCAGATAAATAAATAGCTTTTGAGCACTGAAAAAAAAGCTCCATGAtttgttgtgacttgcggtgtatggtaaggtgattcattggcggaattgggaccaaaagctagagttcatgctttagcttagtggccttagactttttgtaatgagaacaaaaatatgacagtatctctttatatggtggacatacatgggaaatgtcctcagggttgcattttaattcctcagagtttctgttataggactggctggacagtggctcagaataagctatttaacaagatcctcaaagccctgcagtctgaccggcttgcacgattggccaatgaaggggtaagacattggaagtctgtctatagttgactctcttagttttggcctcgggggttgttgtgtcctgtgtgctctctcccctcagcccccagcttgctcacggagcgtaactcttgttttattctttttgtgaacacctttcaggcttgcaatgagcctgtgctgcgtcgcgtggctgtggacaagtgtgcaaggagggtgcggcaggcgctggcaagtgtcagctgggacaccaagctgacccagtggctgcacaccaccctcgtggagaccctgagcctgcccatgctggctgcttacttggatgccctgcagacactgaaagggaaggtaagactgtgcttggtttgcccgagtggcagcagccagggtttgctgtgagttcagagggtttgcacaaggccctgtgactaggtcagcacagaagctaacaatttggctattataatggaaaaggggatcattttcccttctatttggtataaatttcttcagtcgtgccatttttttggataaagtgtggtatctgaaaaccaggatacataatttccctctatagccttgtagttcttaccttgagtcatcgctgacttgaactgtggtggaagagACGAGGCTGAGCAGAGGAGGACCAAGGGTACCTTCTGAGTTAACTCTCCTCATGAAGAGATGGACTGTAGGCTGAGTATGGTGCTACACACCTTTAACCACAGAACTGTACAGGCAGAGGCAGGTGGCTCTCCATCACTTGGAGCTTGGTCTACACTGCAAGT

This mutation is an 898 bp deletion beginning at Chromosome 1 position 36,357,760 bp and ending after 36,358,657 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43995 TTT CCC TCT ATA GCC TTG TAG TTC Wild type Forward A
43996 CAG AAG GTA CCC TTG GTC CTC Common A
43997 TGT GAT TAT TTT AGC CAC CAG A Mutant Forward A
43998 Fluorophore-1 CTT GAG TCA TCG CTG ACT TGA A Quencher-1 WT Probe
43999 Fluorophore-2 AAA GCT CCA TGA ACG AGG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43995 0.40 uM
43996 0.40 uM
43997 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.