Protocol 31864: Probe Assay - Gpr135<em1(IMPC)J> Probe Alt 1
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

>chr12:72069605-72069705 101bp TGTGCTCTTCTTCCGAGAGG ACCCCTGCCCTTCTTAAA

Mutant=  83 bp

Wild Type = 101 bp

Sequence

Wt Sequence: tgtgctcttcttccgagagggtccaccggactcagttatggaagtcggcaaactgcataattctgaaacCAgggacagcagcatttaagaagggc

 

Mutant Sequence: acgtcaaggttccaccagtcccgagtggtgaccgagtcccacccactccccgcagctgccgttctggtccgcacaacctttcaccctgctgcgggatgagcagagcggcggCAgggacagcagcatttaagaagggc

1370 bp deletion beginning at Chromosome 12 negative strand position 72071005 bp and ending after 72069636 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
36666 TGT GCT CTT CTT CCG AGA GG Wild type Forward A
36668 Fluorophore-1 ACC GGA CTC AGT TAT GGA AGT C Quencher-1 WT Probe
37860 GTT CTG GTC CGC ACA ACC Mutant Forward A
37861 ACC CCT GCC CTT CTT AAA TG Common A
37862 Fluorophore-2 CTG CGG GAT GAG CAG AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
36666 0.40 uM
37860 0.40 uM
37861 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.