Protocol 47840: Probe Assay - Klhl35<em1(IMPC)J>-Alt3
Version 4.0

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=  118 bp

Wild Type = 111 bp

>chr7:99470282+99470392 111bp AGCCTGCCTGGTTATACACG GGTGGGTGGGAAACTAGAGG

Sequence

Wt Sequence (deletion in lower case):

AGTCATATTCAAAACTAAGCGTGACTCTGAGAGCCCGTAATCCATAAGCCTCAGTTTTCTCCTCTGTAGAAGGCGCAATAGTCCTAACAACCATCTGAAGGACTAAACAACAAAAGGGGAAGAAAGGCTTTTTCCCCCTGTAACTTAGAGGCCAGCAGACTGGGGTAAGAGTTAGTGACTTCTTCCTGTCTGTAACCAGAGGTCAGGATGGAAGTGGTGGCTTCCAGGGGCAGCTGCCAGGGTAGGCAGAAAGCCAGCGgcattgtctggtgtgatcaaaacagaaaggacctgagttggaagcctgtaaatccagtgccagtgccgagtaagcagaaacaggatagggagttcgagaacagaggctgtagaaaaaacaaacccatgtgctgaggatatagtttagagaagagcacacagcgctctggttcagtaatccccagcaccgtttggggggcgggggcgcacagcgtgagaagaacagcagtgtaggtgtggatctagaggtcttgaatttatccccaccccaggttcatggacttagctgaagtgatcgtggtcattggcgggtgtgaccgaaaggggctcctgaagctgccttttgcggatgcttaccacccagaaagtcagcgctggaccccactgcctagcctgcctggttatacacgctctgaatttgcctcctgtgcacttcgaaatgacatttacgtttctggtgagggtacaagtaaaataggagcctctagtttcccacccacccccttatattttcctccagtgaggagatgggggctggttagtgactaaaactgtgtggtccagcaaagcctagtcaaaatgggagtgctgatgctggggtttgggccgaaggctttaagccttggtgttagtctttgctctgacaagaggctacctggtattcagtcggccattcctggtccctaaactccatcctcatttcctatatggagaccctgtAATAATCCTAATACCTCAGAGAATGAGGATGGAAATGAAGGCAAAGCTTTAGTAAAGAGAGATGACTGTGCCCCGTCCACTCCACCACTGTGCAGACACTCTGCTTTCCCTTGGCCCAACACACATTCTCAGTGACACTTGGGGACACTCCTGCTTTGCTTCCCTGAGCACAAGATGTGTCACACCACAGGC

 

This mutation is a 719 bp deletion beginning at Chromosome 7 position 99,469,893 bp and ending after 99,470,611 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
68450 AGC CTG CCT GGT TAT ACA CG Wild type Forward A
68451 GGT GGG TGG GAA ACT AGA GG Wild type Reverse A
68452 TGT AAC CAG AGG TCA GGA TGG Mutant Forward A
68453 AAG CTT TGC CTT CAT TTC CA Mutant Reverse A
68455 Fluorophore-1 AGG CAG AAA GCC AGC GAA TAA TC Quencher-1 MUT Probe
71038 Fluorophore-2 CAC TTC GAA ATG ACA TTT ACG TTT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
68450 0.40 uM
68451 0.40 uM
68452 0.40 uM
68453 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.