Protocol 36376: Probe Assay - Tmem33<em1(IMPC)J>Alternate1
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

>chr5:67263908+67264042 135bp ATGGGATTAAGACTAACACAAAGC CCATCTCAACCCAAGACATCTAGT

Mut= 133 bp

Wt= 135 bp

Fam=Mut

Hex=Wt

Sequence

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

 TTCTATGCCAACTGTTGCAACGATTTTACACTTCTCTTTTTAACGGATTATTTAATCCAGACCTAATATCTGATGCTTTATTCAGTTCTGAGTCCTGACTATGTGGGTTTGAGATTTTTCTAAGAATAGACATTATAATTCTCTTGAGAGCCATTAGTCATTATTTAAAGTTGTTTTAAAGTAGATGGAGCCATTATTATAAATGTTGAAATAGCACAGAAAGTTTTACTGTCTCGATTTTTTTCTGAAAGTGTTTCTCATGCTTTCCATA^gcatatgtgtgagcaagtggttaagtaacgtgagtttccacttactgatctgcaaaagcaaatattacaagatggtgttacaatattcttcataggattatagtgcataatttgtccttaacatagtttttgggcttagggtaccataatttttttttttttaatctagcaattcatgatgaccaataaattggacacagcaatgtggctttctcgcctgttcacagtttattgctccgctctgttcgttctgcctcttcttgggtatgtattatcttgtttctgactcagaggtgttcggtcagtatctatgactgctttttagctttagaattgttcaattgtaaatgggattaagactaacacaaagcccacttaacagcaacagtaatctggtaataccaattaggatgttttatttctagtttgtgtgactgctcgttgaaaagctga^GGAAAACTAGATGTCTTGGGTTGAGATGGATTCTGCAGGTCTTTTAAGTTGACAAAAGTATGACTGCTTTATACTAATCAGTTGGATGTGTATGTGAAGAGATGTTTCCCAGAGTGTGGAGTACATTTGTGTAATTTGTTTTCTATTGACAATTAAATGATTTAGGTGTTGTGCAATTATGATGCAATTCACAAAGGAGGAAGAGACGTGGTAGCTGTTGAAGTCTCAGAATAACTTTTCCTCTGGTAATTTGCAGGTTGCATGAAGCAGCGAGCTTTTACCAGCGTG

This mutation is a 453 bp deletion beginning at Chromosome 5 position 67,263,561 bp and ending after 67,264,013 bp (GRCm38/mm10). In addition, there is a single bp (A) insert at the deletion site.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47830 ATG GGA TTA AGA CTA ACA CAA AGC Wild type Forward A Wt F
47831 CCA TCT CAA CCC AAG ACA TCT AGT Common A Common
47832 AGT TGT TTT AAA GTA GAT GGA GCC A Mutant Forward A Mut F
47833 Fluorophore-1 AGT TTG TGT GAC TGC TCG TTG A Quencher-1 WT Probe
48566 Fluorophore-2 AGT GTT TCT CAT GCT TTC CAT AAG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47830 0.40 uM
47831 0.40 uM
47832 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.