Protocol 33714: Probe Assay - Zfp512<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

>chr5:31465595+31465754 160bp TGAGAAGAATCAAGCCTGCTG TGCCACGATAACCCATTACC

Mut= 160 bp

Wt= 160 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AACCTAATCACTGTAATCCTTCATAGGCACGTCTAGGCTAATCTAATCTAGAAGGTTCCTCACAGGTATGCCCTCCCTTGATTCTCCAGCCTCTTGAATGTAAACTCTGAGTAGTCTGGGAAGGCCTGGGCTGCAGAGGGATCAGTTGTGTAGCCTGCTTGCTCTGACTGCCCTGGGTgtaagaggaattgtgtacactgtgttgaattgacttgtaattgttagtatgttttaacccatgtacttttttttgtatgtggtttctttttgttttatttatttatttatttatttatttatttatttatttattttgctttttgtaaacaaaacaaagaccagtcttcatagttttacacaagttattagcttggttgtatttttacaatatgagaaaaattagtaggaaatgtctccagcttctcctgtgtggtccatgactttgtgttgtatcagtaatagggcccagtgctccataaaggataacagtttccagtacaccatccctcatgaggactccctgagtggctcgtcatccgcctcctcttgtgaaccagtcagtgacttcacagcaaccctccgaaaatctacctactggatgaagatgagaagaatcaagcctgctgccacttcacaggttgaaggtatccatttcagattctcagctctgagacaggagagtgtatctagtgtgcttgatattttggtatggtatggttgtttatcagtagtgaggtgagggctgggtaatgggttatcgtggcaatgtatgccagagagtcaccatcagtggttgatttatagctgtctttgttgagtgctgcttctgtagacccatttaagcataaggaagatctaatggtttcTGAGGGAAGAGGGGACTCCATGGATTAAAAACAATTCACACCAGTGGTGGCACACGCCTTTAATCCCAGCACTTGGGAGGCAGAGGCAGGTGGATTTCTGAGTTTGATGCCAGCCTGGTCTACAAAGTGAGTTCCA

This mutation is a 688 bp deletion beginning at Chromosome 5 position 31,465,168 bp and ending after 31,465,855 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42045 TGA GAA GAA TCA AGC CTG CTG Wild type Forward A
42046 TGC CAC GAT AAC CCA TTA CC Wild type Reverse A
42047 CTG CAG AGG GAT CAG TTG TG Mutant Forward A
42048 TGG CAT CAA ACT CAG AAA TCC Mutant Reverse A
42049 Fluorophore-1 ACT TCA CAG GTT GAA GGT ATC CA Quencher-1 WT Probe
42050 Fluorophore-2 CCC TGG GTT GAG GGA AGA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42045 0.40 uM
42046 0.40 uM
42047 0.40 uM
42048 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.