Protocol 45658: Probe Assay - Guf1<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

>chr5:69561020+69561134 115bp CAGGTGAGAAAACTCCCCTTA CTGAAAGTGTGTAGAAACCAAAGC

Mutant=  84 bp

Wild Type = 115 bp

Sequence

Wt Sequence: caggtgagaaaactccccttatattgtgagaatatacagaatcattattaCagaggccccaggaaggaagagtgtagccttaaatctcaatgctttggtttctacacactttcag

Mut Sequence:  caggtgagaaaactccccttatattgtgagaatatacagaatcattattaCTatggggctgaagtacactggcatttgtttgcg

A 377 bp deletion beginning at Chromosome 5 position 69,561,071 bp and ending after 69,561,447 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66159 Fluorophore-1 CCC CAG GAA GGA AGA GTG TAG Quencher-1 WT Probe
66160 CAG GTG AGA AAA CTC CCC TTA Common A
66161 CTG AAA GTG TGT AGA AAC CAA AGC Wild type Reverse A
66162 CGC AAA CAA ATG CCA GTG TA Mutant Reverse A
66163 Fluorophore-2 TAC AGA ATC ATT ATT ACT ATG GGG CTG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66160 0.40 uM
66161 0.40 uM
66162 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.