Protocol 35707: Probe Assay - Zfp786<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

>chr6:47827130-47827240 111bp TCTCTCCAGCCCACAAGTCA CGAAAGACCATCTCATTGTTCA

Mut= 118 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

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

 GTCTGTATATCACATCTGTGCAATGCCTCTGGAGAGCAGACAAGGGGGCACTAGGTTCTCTAGGACTAGAGTCAGTGATGGCTGTGAGCTACCATCCGAGAGTTGGGCATGCTGAGCCATCTCTCCAGCCCACAAGTCACTTTTGCAGAAGAGTCTGCTGAGTTAAAAACAG^ctgtccactctgtgagtctcaaattacttatgagagtgaacaatgagatggtctttcgtagccgtcatgacggtatctgtatgatgtctttcacttcagttcccactgacttttgaagatgtagccatttacttctctgagcaagagtggcaacacttagaggcctggcagaaggaactttacaagcaagtcatgagaaccaattacgagactctcatctctctaggtaaggagctttcaacttctctggtgattcttgcaactggatcttctcagagccccttggtcacttgatacacctgttaggtagcaagattgcttagatataacccagaaaggctttttcatctctttattgttgttgttaatactaatgtatacatcaccggtttgtccttagaggt^GGACCTTACAGGGAAGTATTTAAGACAGCATGTAGTATCATAGTCAGAGGTAGCAAGGATGAATGTTAAAACTTTCCCCATCACACTTTACTCTGAGATTGCCTGATTTTTCCACTGAGAAGCACACCTGCCTGTGCCCATAAAGTACCTGTATGCCTTAACCTAGAAAGTGGCTTCTCTGCA

This mutation is a 404 bp deletion beginning at Chromosome 6 position 47,826,784 bp and ending after 47,827,187 bp (GRCm38/mm10). In addition, there is a single bp (A) insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46812 TCT CTC CAG CCC ACA AGT CA Common A
46813 CGA AAG ACC ATC TCA TTG TTC A Wild type Reverse A
46814 CAT TCA TCC TTG CTA CCT CTG Mutant Reverse A
46815 Fluorophore-1 CTG TCC ACT CTG TGA GTC TCA AAT Quencher-1 WT Probe
46817 Fluorophore-2 AAA CAG AGG ACC TTA CAG GGA AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46812 0.40 uM
46813 0.40 uM
46814 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.