Protocol 37256: Probe Assay - Pjvk<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

>chr2:76652261+76652355 95bp GTCATGGAGAGCATCCGAAC AAGCCCTGAGTGTATCTTACCC

Mut = 96 bp

Wt = 95 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

TAGAAAACTAAACAAGTCGTGCCTCCACTCTATAGAGCAAGAAAAACAACTGAAGAGAGAGGCAAATTAGTCAAGTGATTTTGTTTCTATTTTCTGATTTTCATTATTCCTACctcagattcacacacgtcattaagatgggccttaaataacacaaaaaggtaccttcagtcacatgatcacatattagagcatcttagacacatgtctttttttattctagaaaaattaactttgaccacagtttgatccgccaatcaaggagcagcaggaaggcggtgctgtgcgtggtcatggagagcatccgaaccacacgccagtgctctctgtcagtgcacgctgggattcgtggggaagccatgcgggtaagatacactcagggcttcactaacgacacagttactatggagtttaaaaaaaaattactcatcaaaggcagaaaaaaaaaaaaaaaaaaagagcctcagttttgtagggcttcagtccggagtggctgtgctttggaataagaaaaccaaccaaaaccacttgggttctaaccctgtgtgctacggtacaaacaatatattactggagtctctccaattaactactacctcagtgttagagtaacCACATAAAGTAAGCCTTGATGTCGTATGCTTTGACATTTATTTATTTATTTATTTATTTATTGTTTTTTGAGAAAGGGTTTCTCTGTGTAGCCCTAGGCTATCCTGG

This mutation is a 500 bp deletion beginning at Chromosome 2 position 76,652,084 bp and ending after 76,652,583 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50905 GTC ATG GAG AGC ATC CGA AC Wild type Forward A
50906 AAG CCC TGA GTG TAT CTT ACC C Wild type Reverse A
50907 CTG AAG AGA GAG GCA AAT TAG TCA Mutant Forward A
50908 AAA GCA TAC GAC ATC AAG GCT TAC T Mutant Reverse A
50909 Fluorophore-1 CTC TGT CAG TGC ACG CTG G Quencher-1 WT Probe
50910 Fluorophore-2 TTT CTG ATT TTC ATT ATT CCT ACC ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50905 0.40 uM
50906 0.40 uM
50907 0.40 uM
50908 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.