Protocol 31344: Probe Assay - Efcab7<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr4:99877844+99877947 104bp CCTCTCCTGGTGTACCATTC ATTTTCCTGAGCCAGACTGC

Mutant=  139 bp

Wild Type = 104 bp

Sequence

Wt Sequence: cctctcctggtgtaccattcattttgactcAGagtgtgacctcactgattgatggctacgtttcactaggtcacttggggctgggcagtctggctcaggaaaat

 

Mutant Sequence: cctctcctggtgtaccattcattttgactcAAAGGGGGAaggtcactgagaatccaagaattagtcaccatttttagactttcctagcagtaaaaatctactagagtaaaatttttattgtgggattttacttggctagg

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36661 CCT CTC CTG GTG TAC CAT TC Common A
36662 ATT TTC CTG AGC CAG ACT GC Wild type Reverse A
36663 CCT AGC CAA GTA AAA TCC CAC A Mutant Reverse A
36664 Fluorophore-1 TGT GAC CTC ACT GAT TGA TGG Quencher-1 WT Probe
36665 Fluorophore-2 AGG TCA CTG AGA ATC CAA GAA TTA GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36661 0.40 uM
36662 0.40 uM
36663 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.