Protocol 34150: Probe Assay - Tesk1<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

>chr4:43443887+43443976 90bp GCACCCTAACATCCTCAGGT CATCTCCCTCCTTTCCCAAG

Mut= 88 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case; and insertions with carrots ^g^ (C insertion):

 GGCGGGACCGAGCCTTGGACGGGAGGCTAGAACTTAGAGGCCCCGCAGCCTGGGTTAGAGATCCTTGAGCTAGCAGCCACccaacCTGTCGGGCACACACGGAGCCTTGCCTCCCCCAGGTTCGGCACCGACAGTCGGGACAAGTCATGGTGCTGAAGATGAACAAGCTCCCCAGTAACCGGAGCAACACGCTAAGGGAGGTGCAGCTGATGAACCGGCTCCGGCACCCTAACATCCTCAGGTGAACGGCCTAGCCTCTT^gctggacagcttgctgggcaggaggaaaagtcccttgggaaaggagggagatgcagtggagcccgggctcttacagcgtatcctttatcctccccatcacctttcaggttcatgggggtctgtgtgcaccaagggcagctgcacgcgcttacagaggtaagcattaagcataggccaggaaagagggaaggcaccacacccccaagtgagagtgtcagtgatcttggtgggtctgctgggaaattaaaaagaagccacctgcctgcccaggacctcaggacctcctccaactaaaggcttcctgaacttccctgtgtgcaaaccacttattagaatactcagctggagttagtgatggagatattttgtatctgggtccaatacaggagccactagctacacgtggaatagactaaaacaagagctggtgtaacaaaagaatgtctgattatatttaaatgtattggataacatagctgtacatagtcctactgtgagagacagggtagcctagtatataaatgggaaccttagagcacagacccaccaacccttttgacctttgtgagcctcctgagtgtcaactgtcttaggagtggcaggcacagagcaccgacattcacacaccttgtctttccccatgtcccagtatatgaatgggggcaccctggaacagctgctcagctccccagaacccctctcctggccagtcaggctccacctagccctggacattgcacaaggcctacggtacctacacgccaaaggtgtgtttcaccgagacctcacatccaaggtaggctggccaagacgggtggaacgattaaagagggtttgtgactgttaagttgtaattggcctgtggatattgacatgtaggtagtagatacgttaggatg^TCGGGGTGCCAAGGTGTTGGAAACTATTAAAGAATGGGGACAGCAAGAGGGCTGGGGAATTGAGTACTGAGTGAGGTGGTGATAATGAGGTGCATTGGAGAACTGGGAGGGAAGGT

This mutation is a 899 bp deletion beginning at Chromosome 4 position 43,443,924 bp and ending after 43,444,822 bp (GRCm38/mm10). In addition, there is a 5 bp deletion (CCAAC) 175 bp before the deletion and a single bp [C] insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42934 GCA CCC TAA CAT CCT CAG GT Common A
42935 CAT CTC CCT CCT TTC CCA AG Wild type Reverse A
42936 CCT CTT GCT GTC CCC ATT CT Mutant Reverse A
42937 Fluorophore-1 TTG CTG GGC AGG AGG A Quencher-1 WT Probe
42938 Fluorophore-2 CTC TTC TCG GGG TGC CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42934 0.40 uM
42935 0.40 uM
42936 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.