Protocol 34118: Probe Assay - Kctd11<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

>chr11:69879468-69879560 93bp CCCTGAAGACCTTCTGAACTC CTGACTCATCCCCCTCCTCT

Mut= 84 bp

Wt= 93 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCTGAACGTTACCACCCAATTCTTTCCAAACCCCCAAATATCTGCTTTTTCCTTCTCCTTGCCTGTCTCTTATATTACCCAAAATTTCCCCCCCTCCTGTGTCCTCTTTACCCCCTTCCTTTGGGGGTCCCGTGACCCTAAATGTGGGGGGAACACTATATTCCACTACACTGGAGACCCTGACTCGCTTCCCAGATTCCATGCTGGGGGCCatgtttagggctgacaccctaatgccagccaaccttaacccgcaaggagatggccattacttcatcgacagggatggcaaggctttccggcacatcctcaattttttgcggctaggccgtctggacctgccccgtgggtacggagaaactgcccttcttaaggcagaggctgacttctaccagatccggcccctcctggatgccctgcgggaattggaagcctctcggggtacacctgcatccacagccgccctactccatgcagatgtagatgtcagcccccgccaggtgcacttctccgctcgaaggggcccccaccactatgagctgagctctgtccaggtggacaccttccgagccaacctcttctgcactgaccctgagtgtctggctgccatgcgcaacagatttggtgtggccattggggacagggcagaaggaggtccacattttcggctagagtgggcctcccgcccccaggaactccctgaagtagagtatcaaagactggggctgcagccactgtggactggggggccagaagatcgtcgggaggtagcgaacacgcctacattcctggaggaggtgctacgggtggctctggaacatggcttccgcctggactctgtcttcccagaccctgaagaccttctgaactctagatccttgcgctttgtgcgccactgagggtgctgccCTCCACTTGACTGCAGAGGAGGGGGATGAGTCAGCCAAGGGGCCTGTGGCTCTTCTAAAACCTCTTTCTAGTACTGGTGTGGTAGCTATGAAAGTCAGGGATTCCACCATACCTGGCCAGAGCCTAGAGCTGGGGTTCCCAGATCGAGCTGCCCCCTTCCACCCCACAAGACTCACGAATGGT

This mutation is a 697 bp deletion beginning at Chromosome 11 position 69,879,502 bp and ending after 69,880,198 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42867 CCC TGA AGA CCT TCT GAA CTC Wild type Forward A
42868 CTG ACT CAT CCC CCT CCT CT Common A
42869 CCA CTA CAC TGG AGA CCC TGA Mutant Forward A
42870 Fluorophore-1 CGC TTT GTG CGC CAC T Quencher-1 WT Probe
42871 Fluorophore-2 TGG GGG CCC TCC ACT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42867 0.40 uM
42868 0.40 uM
42869 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.