Protocol 37508: Probe Assay - Cog8<em1(IMPC)J> Alternate2
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

Mutant=  119 bp

Wild Type = 119 bp

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): CTCCCAAGTGCTGGGATTAAAGGCGTGTGCTACGACCGCCCAGCGGGAAGATTTTAAATAAACTAGGTAAACCATCTAGCCCGTGTCtagtaagtgcttggtaacaaactgctgtgtaggataataaggaaatacactttcctgatcggctctaggaactttgtgaaggaagctgaggagatcagctccagccgccgcatgaacacgctgactctaaaccggcacacagaaatcctggagattttagagataccccagcttatggacacgtgtgtccgaaacagctatcatgaagaggcgctggagcttgccgcctatgtacggcgactggaaaggaagtactcctccatccccgttatccaggtagccaccttgcccaggGGCCTCAAGCTGATGATCTGATAGTTAATTCTGTGATTGTGACTACACTTACACGTAGAAACCTTTGCATAGTTCATATCTTTCTGCCAGCCAGGTAAGCTG

 

Mutation details

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49597 GCC CAG CGG GAA GAT TTT A Common A
49598 CCT AGA GCC GAT CAG GAA AG Wild type Reverse A
49599 TGC AAA GGT TTC TAC GTG TAA G Mutant Reverse A
51514 Fluorophore-1 CCG TGT CGG CCT CAA GCT Quencher-1 MUT Probe
51515 Fluorophore-2 AGC CCG TGT CTA GTA AGT GCT TGG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49597 0.40 uM
49598 0.40 uM
49599 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.