Protocol 34814: Probe Assay - Zbtb44<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

>chr9:31064317+31064437 121bp AAAACAGCACATGCTTATCCAC AAGCAGGATTGCAAAGCACA

Mut= 123 bp

Wt= 121 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case and insertion with carrots ^g^ (ACTGTAGGAACTGATGAGAGTTG insertion):

 TGGACTGGGAAGAGAAAATTCCTATATGAGTAGTTTTAGACACTTTTTGTGGGAGTTGAGAATTTATTCTACAAGAAGACAGTTGTCACAGGTTTTAAAGGGTGAAATAAGGGTCTAAGTTAAAGCAGAAGAAAGGAGGGAATTAAAGAGGTGAAGGGCATCTAAAAAGC^agcctgggatctgttttgtgttttatgtccacactaaagatgctggacatttagacaattacattgctttatacatttttctaagtgaaaatctgtttttaaaagtctttgttgcttcctttgttcatttaagagttggactacagaaatgtgtattcaacttaaacatcagagcctcgttaccttctttggtttatttttttagattggaaaatgttcagtatccctatcaactctacattgctccttctaccagcagtacagagcggccaagtccaaatggtccagacagaccgtttcagtgtccaacctgtggggtccgcttcacccgtattcagaatctaaaacagcacatgcttatccactcaggtaatgtcaccttcagggttgttgtatgggggggg^GGGGCGGCACGGGGGGTTGCAGGCAGGCAGGCAAAAACGTTGTGCTTTGCAATCCTGCTTAAAATACAAAAGACTTTGTGCCAGGCCAGACATAGTGCCAAGTGCCTTTAATCCCACCACTCAAGTCTAAGGCAGGCAGATCTAGAATCTGAGGCCAGCC

This mutation is a 404 bp deletion beginning at Chromosome 9 position 31,063,974 bp and ending after 31,064,377 bp (GRCm38/mm10). In addition, there is a 23 bp insertion (ACTGTAGGAACTGATGAGAGTTG) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44535 AAA ACA GCA CAT GCT TAT CCA C Wild type Forward A
44536 AAG CAG GAT TGC AAA GCA CA Common A
44537 GAA AGG AGG GAA TTA AAG AGG TG Mutant Forward A
44538 Fluorophore-1 ATG TCA CCT TCA GGG TTG TTG TA Quencher-1 WT Probe
44539 Fluorophore-2 CTC ATC AGT TCC TAC AGT GGG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44535 0.40 uM
44536 0.40 uM
44537 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.