Protocol 45385: Probe Assay - Gtf2a1l<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

>chr17:88689733+88689833 101bp GAAGTCTTTAAGGTTGCCAGGA TCAATAGGTCATGTTAGACCAAGC

Mutant=  102 bp

Wild Type = 101 bp

Sequence

Wt Sequence: gaagtctttaaggttgccaggaaatgtatatagttcaatactttccCctcagatggagccagtaagtttagaatttagcttggtctaacatgacctattga

Mut Sequence:  gaagtctttaaggttgccaggaaatgtatatagttcaatactttccCCtcagtgcagctatgcatcagagtgaggacattcctgacactgtagcatgtggga

A 406 bp deletion beginning at Chromosome 17 position 88,689,780 bp and ending after 88,690,185 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
65482 GAA GTC TTT AAG GTT GCC AGG A Common A
65483 TCA ATA GGT CAT GTT AGA CCA AGC Wild type Reverse A
65484 TCC CAC ATG CTA CAG TGT CA Mutant Reverse A
65485 Fluorophore-1 TAC TTT CCC CTC AGT GCA GCT A Quencher-1 MUT Probe
65486 Fluorophore-2 CCT CAG ATG GAG CCA GTA AGT T Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
65482 0.40 uM
65483 0.40 uM
65484 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.