Protocol 42015: Probe Assay - Ttc17<em1(IMPC)J> Alt 1
Version 2.0

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=  133 bp

Wild Type = 116 bp

chr2:94376025-94376140 116bp GAATGCTTCCTCCTGGGTACT CTTACCTGGAAGAGAAGTGAAGC

Sequence

Wt Sequence (deletions in lower case)

AAAGTCATGGTGATCCTCCTGCCTCAGTCTTCCACCATGGCGCTATAAGGATCTTTGGAGCAGTGCAGTGGTTTTCGGCTGTATCCCCGTGTTCTTATTGCTTTCTTTATCTTGAGTTTCAGAAGAGCCAGTGTCTTAATTGTGCAGATTTTGAAATCCTCAGTCAAAAAAAACAAACCTGTCtttgattatacttgactatttttttgtttctctctagaatgcttcctcctgggtactgtataacctggcttcattttactggagaataaaaaatgagccatatcaggtagtggagtgtgccatgcgagcgcttcacttctcttccaggtaagatttctcttcttcgggcctccagatcagccgcaccaatgaggactgttaaagagtaacacataggaaaccacaaaaagcagtgttgtcttgtcgtcacacacttctccgtcTCTTAATCAGAGACATCATTAGTCTTGTCAAGAGAAAGGATCACGGGTGTCAGTAAGTTCAAATTGAAAGCTGTGTACCTAACATTCATTAAGGAGAAAAATACGTAAGGTAGATAGGCTGTCGTTCACCTGAGATGTTAGTGCCAGGACATACACTGGAGGAATTAGCAAGAGTTGAGCCCACCTTTCCTTGG

 

This is a  263 bp deletion beginning at Chromosome 2 position 94,206,259 bp and ending after 94,206,521 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
58510 GAA TGC TTC CTC CTG GGT ACT Wild type Forward A
58511 CTT ACC TGG AAG AGA AGT GAA GC Wild type Reverse A
58512 Fluorophore-1 ATG AGC CAT ATC AGG TAG TGG AGT G Quencher-1 WT Probe
58513 TTG AGT TTC AGA AGA GCC AGT G Mutant Forward A
58514 TGA ACT TAC TGA CAC CCG TGA Mutant Reverse A
59677 Fluorophore-2 ACA AAC CTG TCT CTT AAT CAG AGA CAT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
58510 0.40 uM
58511 0.40 uM
58513 0.40 uM
58514 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.