Protocol 34485: Probe Assay - Zfp787<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

>chr7:6131056-6131169 114bp TGCATCTTGATGGCTGCT CCATGGTAACTGCCAACACC

Mut= 123 bp

Wt= 114 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCCTAGCCTGCAGACCTGGCCCTCCCATCCTCACTGGGTTTCAGGTGCCACCTGTGTTGTGCTTCTCATTGCCCTCCTACCCTTGGCCCTCGATAGCATTGGTCGGTAGACCTTTATGCAGGCCTGTAGCCTTAGGCATTGGTCTGCAGATCTAgagaaaggctaccagagacaacacccactcggaaggagatcaagtattagggagatggattatgaacacagaaatacttgaaatgccttgtgtggttggtgtcacacgggataaggtgtactggatatgcattggtacaacccagcagtccccaggttggaggaaccccgaaaggaagagctgccctgggccactcaataagctggaatccattggcagcctgcgcatggatgttcacctctgacttgcctcctccttccctcttgctttgcagtggacatcctcatcatggatgacgatgacgtccccagctggcccccgaccaagctgtccccaccccagtctgcacctcctcctgggcccccgcccaggccgcggccaccagcaccctacatctgcactgagtgtggcaaaagcttcagccactggtcgaagctgacacgacaccagcgcacgcacacaggcgagcggcccaatgcctgtaccgactgcggcaagaccttctcgcagagctcgcatttggtgcagcaccggcgcatccacacaggcgagaagccatatgcctgctcagagtgcggcaagcgctttagctggagctccaacctcatgcagcaccagcgcatccacacgggcgagaagccttatacttgccccgactgcggccgcagcttcacacagagcaagagcctagccaagcaccggcgctcccacagtggtttaaagcccttcgtgtgtccgcgctgcggccgtggcttcagccagcccaagagcctggcacgccacctgcggttgcacccagagctgtcagggccaggtgtggcagccaaggtgctggcagccagcgtgcggcgcgccaaggcaccggaggaggccacagcagcggacggcgaaatcgctatcccagtgggcgatggcgagggcattattgtggtgggcccgccaggggatggtgctgcagcggccgcagctctggctggggtggggacgcgggcaacggggacacggtcgcgcagggcgccggcgcccaagccgtatgtgtgcatggagtgcgggaagggttttgggcacggggctgggctgttggcccaccagcgcgctcagcacggggatgggctcggggtagcggttggtgaggagcctgcacacatctgcgtggagtgtggtgagggctttgtgcagggtgccgcactgcggagacacaagaagatccatgcagttggggcgccttcggtctgcagcagctgcggacagagtttctatcgggcgggtggagaggacgatggagaggaccagtcggctggtgcgcggtgtgccgaatgtcggggtggggaggcccggtagggacggggaggcccggggggtggacagggcccctcgggctcggtgggaactaccagtgtgcaagaccacgagggaggcccagagagaatcagaccgaaaggaacagcggccttgtctccttccttactccgtgctgttctgggtgtgaggggacctgcaactattgggttctcaggactgtccacagcctctctgccgacccgccctaccgcctggtgggctggccctttgtaggtttccattctgggagccagagactgagagaactgcaaggatcaattggcactagaccctcaagggaaagaccacccccactacaggtgttgggctgcggtgtcgtaagaaatggagggaatcctggccttatgcatcaaaagggcttccttcccttctagaattttcttttaatcatactgtctgacccgtcccatccattattttttctgccccccatgggtccttatcaattttctagcccaagtcgttcctcttcctcttggatttgcagtagcagaggcgtgaagtagcctcctgttcccagcacggtcttattgagaggtcctggcagtggaaaaagggatgtgggggcaataaatggcattattcaattgtcccagcactgcctgtttcttgagtttgggatggagaagggagagaggttaaacctagccatcctcactttggtgcacacaagtgacaagcaccagatgatgggatgaccatgttggggggcccagagtgtcccaagccagaaagagatgtgtttgccaaggagccccttgctgacctggtacaaggtcactgttgggaaggtagtctgttcctgctgggaaaaggatggcttatggaagacatagaaatagcacacccctctatctctggtccaagttctggtcactcatcagccatatatcagcattctctgcagagcaggctgcatcttgatggctgctactgttcagtgaaggggcggaggcggaggaggaggagGAGCTTGGAGTAGATTGTTTCTGGACCCAATACTGAAATGGTGTTGGCAGTTACCATGGCATGCTTTGTTCCAGATGCTCCTCAGTGCCAGTCCAGTGCATCCCCCAGAACCCTACAGCTGCAGGATCAGAAGGGACCTCCAGCGTTAGGTGTA

This mutation is a 2331 bp deletion beginning at Chromosome 7 position 6,131,115 bp and ending after 6,133,445 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42657 TGC ATC TTG ATG GCT GCT Wild type Forward A
42658 CCA TGG TAA CTG CCA ACA CC Common A
42659 CGA TAG CAT TGG TCG GTA GA Mutant Forward A
42660 Fluorophore-1 CTG TTC AGT GAA GGG GCG Quencher-1 WT Probe
43740 Fluorophore-2 TGG TCT GCA GAT CTA GAG CTT GGA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42657 0.40 uM
42658 0.40 uM
42659 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.