Protocol 44053: Probe Assay - Ercc8<em1(IMPC)H>
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

>chr13:108168972+108169122 151bp GGCAGTGTGTGGAACAGTCC GCATTCAGTGTGTGTCTGTGTG

Mut= 168 bp

Wt= 151 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence:

gcaaccaggtagagtttgaattcttagctgaaaagaacaaaggtgtttatgaactgctgtccacctgtctgtcttcacccaactgtgggtaaatactgttgtgtggaatcccatggcagtgtgtggaacagtccacagttctaataatgataattgagaaaggaaaatcccacaccggtacagaaaaccactaccacctccaagatgaaggaaacccactgtagttggccacacatacacacgcacacagacacacactgaatgcagtatagtgagcatatcccacatgatgtgcctcaggaaaaggcaagcctccacagctcggaatgggtaccaaatttggagatgcctagatatcctctcttcccttcagagcagctaaaacaaggatgtgtgatcctactgtagaa

Mutant Sequence:

caataaaacttagcacacagctttgccattggattgccttagccaaggatgaggaaaagacaatgtcgagggtctctcactttctataacagaaagtaggtacaaaacagaggctccctgcaaccaggtagagtttgaattcttagctgaaaagaacaaaggtgtttatgaactgctgtccacctgtctgtcttcacccaactgtgggtaaatactgttgtgtggaatcccatggcagtgtgtggaacagtccacagttctaataatgataattgagaaaggaaaatcccacaggaaaagaacgtttttaaagtatagtaaaaatatagatagcagatatgagtattagatgagaattagagaaatttttcagtactactcctttctctcagctttctacctacatacctgtaatacctctctataggaatgtgacatccatcgcttcagagttgtttctcatggtcctgtattctgtgcctgccagagttatgtagcaagaccctttctcaaacacacacacacccacacacacacacacacacacataccagaaaccaacaaaacaaaacccaacatatgtggtaaagattaaatgt

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64660 GGC AGT GTG TGG AAC AGT CC Common A
64661 GCA TTC AGT GTG TGT CTG TGT G Wild type Reverse A
64662 GGT AGA AAG CTG AGA GAA AGG AG Mutant Reverse A
64663 Fluorophore-1 AAA CCC ACT GTA GTT GGC CAC A Quencher-1 WT Probe
64664 Fluorophore-2 AGG AAA ATC CCA CAG GAA AAG AAC G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64660 0.40 uM
64661 0.40 uM
64662 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.