Protocol 33548: Probe Assay - Zer1<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

>chr2:30107303-30107406 104bp ACTCTCCTCGCACAGTTGCT CAGAGGTAGGATGGCCACAG

Mut= 116 bp

Wt= 104 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

AACAGAAAGAGGAGCTTATTCTTGCTTGTCAGTGAAATGTGTCTGTGGTATAATGGCAGGGGACCTTTCAAAGGTACTGGCAGTGGTGGCAGCTCCCAGTCCATGTTCTACAGTACGCCCTCCCACCCTTCcatcaagaagtgagagcagggaatgcagctcaagtggtacaaagctctgactttgatcaaagcaggcagagaagcatgtgcctgtatcccagcacttgagaggtggggtaggaggaccaggggttgaaggtcaggctcagctccacagtgggttgggcagggacaggatgagattagaagccgttgtttctctatgggtggagttcctggtccatgggggagcccaggctaggtccagtcaggtggctagctctgacatgctctttccccagcaccgagccatccaagagcagcatcatgcccttccgggctctgaagaggccactgcagttccttgggctctttgagacctcattgtgtcgtcttacgcacattccagcctacaaagtaagaggtggagttgggaggaggaagtggccaggtaccgcatagcactgagtctggggtcccattacctgggttgtcctgacatgttctgtcactaggtgagtggtgacaaaaatgaagagcaggtgctgaacgccatcgaggcctacacagagcaccggcccgagatcacatccagggccatcaacctgctgtttgacattgcacgcattgaacgctgcaaccagcttctgcgggccctgaaggtcagctcagaacccaaggccctcaggctcccagatactgctgccctccttctctgtccttctgcacccagccctgatcccaggggaacaggtagtcttgccttgcaaacctccgccctttggcagtgctggcctcactgccagcctgtccaggccctgccctagtttctgatctcacatctccccaggagagcaagtctaaggtcagcctttccccagagaagacacggtgacttaaagagcccaggctgtctttgcctgcctctggcagtgccccagctccagccccttccctgtgttcacacagctggtcatcacagccctcaagtgtcacaagtatgacaaaaacattcaagtgaccggcagtgctgccctcttctacctgaccaactccgagtaccgctcggagcagagcgtcaagttgcggcggcaggtcatccaggtggtgctgaatggcatggagtcctaccaggaggtgacggtgagcccctgcctgctgcggcctgcatgcttaggcccagtatcccttcctgctccccaggctccccaggcttagggggcctttgccaactctcctcgcacagttgctgtcccagaaacctgcccctggcacacagggaccgcagaagctggcttcAACTGCAGTCCCCTGCCTGTGGCCATCCTACCTCTGCCCTCTGTGTTCCTGATTTGCCATTTTCTCTGCCCCTCTCTCCCTATCATTGTTGGGGGTGGGGGAGTT

This mutation is a 1269 bp deletion beginning at Chromosome 2 position 30,108,607 bp and ending after 30,108,607 bp (GRCm38/mm10).

Designed assuming this was supposed to be from 30,107,338 bp and ending after 30,108,607 bp. - DSW 7/27/18

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41674 ACT CTC CTC GCA CAG TTG CT Wild type Forward A
41675 CAG AGG TAG GAT GGC CAC AG Common A
41676 AAT GGC AGG GGA CCT TTC Mutant Forward A
41677 Fluorophore-1 CCC AGA AAC CTG CCC C Quencher-1 WT Probe
41678 Fluorophore-2 CCA CCC TTC AAC TGC AGT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41674 0.40 uM
41675 0.40 uM
41676 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.