Protocol 35875: Probe Assay - Zfp507<em1(IMPC)J>Alternate2
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

>chr7:35794645-35794735 91bp TGTCATTGCTATTGGGGACA AGATGAAGACGGTGGGTCAG

 

Mutant=  133 bp

Wild Type = 91 bp

Sequence

Wt sequence with deleted region in lower case

CAAAGTACTTCCCAAAATTCAGTATTCCTTATAGTAGGATGTGAATATATTTTCAGAATTTCAAAAATTTGAGCAGAGAAGTGTGATTTTTCGTTATCTACAGTTAAAATTTGGCATAAGAAATAGTTTATCCAGAGTTACATGAAATCATGTTGGCTTCACAGACTGATGCTTTCCAGTAGCTCGAGGTGACTCCACAGGCCCCAAGGATGGCTGTTTCTTTAAACAAAGAATCATGTCTCTTTAGTTCTAGAATCTCAAGGGTTTCTTTAAACAAAGAGTCATGTCTCTTTAGTTCTAGAATTGCAAGGGATTCaagggacattagttctagaatctcaaggaggctgggtgacaggataaggtgttaaagtcttacccaaggaacagggtctttatctgtctctaggagaccgtttcaaaaaggaattctgagctgtttaagtctattagggacagttgggtgcattgataggaacctcccaacttagaagatatttaaacatttcagtcagacaccagagttgacattatcccataacccatgctctctgctgtgcactgtctctagatatggaagaaagcagcagcattgccatgttggtgcaagagattggagaaccggaggctgtgttaactgctgaaggagtcctcagcccttcctcagaagttgaccaacaaagaaaagctaaagcagatccattggtccacgttatccagaagctaagcaagatagtggggcatgagaagtcacaaaaatgtctactgattgggaaaaagcgcccacgtccaagtgagacagcaaactctcttgaaaagctagagaactgtgagattccagctaaagccactgagtcccctgctgctggggttaggaaaaccgagatgtcacaggcaagttccactcttgcttcaaatgatgggaaggctatgtcctaccagtgtagcctttgtaagttcctgtcaccatccttttctgtgctgaaagagcacgtcaagcagcatgggcagcagcatgacgtgatgctcatgtgctcggagtgccacgccacctccaggagccagcaggagctcgaagcgcacgtggtgagtgagcacgagaacagtgccagtagccaggcccggtccagcccctccggtcagggagccacagaaagaaagagtgagaccatggtagacatcccagtgaacatgggcagtccacagacccatgctgtccagagtgcagccatggcagagtcgggtcggaggaagtggtatgcatatgaacagtatggcatgtaccggtgcttattctgtagctacacctgcggccagcagcggatgttaaagacacatgcttggaaacatgcaggggaagttaactgctcctacccaatctttgagaatgaaaatgagccccttggccttctggcttcttctatgtctgctgcacctggtggggttgatgcagttgtcattgctattggggacagcgagctgagcatccacaatgggccatctgtacaagtacagatttgcagctctgacccaccgtcttcatctcctttagaacaaagcacagaggagggagtacacctaaaccaggcagtcacgctggacgctaacgaggaagaaatgctggaggtgatgtctgattccgaggagaatctctttgctgatagtttgctatcttcagcacagaaaatcattagcagtagcccaaacaaaaaaggtcacgtgaatgtcattgtggaacgtttgccaagtgctgaagaaaccctcccaccaaaacatttcctcattaatgccgaaatggaagaggggaagagcccgagcccctccgaagcccagactgggtgtgtaggagcaggaaatatgtaccatgctgataaatgtactgttgatattggggggttgatcatcggctggagcagtgcagagaagaaagacagtgagctaagtaagggcctggctcctgatgagaatgccccaccaggacggagaaggacaaattctgagtctctcaggctgcactccttagctgcagaagcccttgtcaccatgccgataagagctgctgaactaacaagagccagccttgggcactatggggacataaaccttttagatccagacactggacaaaggcaggtcagtggtccattggcaacatactcaaaaaaaatcatgtctcctcttaagaattctacagatggagtaacaagttttaaccaaagcaattccactgtggtagctctcccagagggaaggcaggaattgtcagatgggcaagttaagacaggcatcagtatgtcccttcttaccgtaatagaaaaactgagagaaaggacagaccagaatgcatcagatgatgacattttgaaagagttgcaggacaatgcccagtgtcagcccaacagcgatgggagtttgttggggtccaacgtggtggagtacatcccagatgccgagcggccctaccgatgccgcctgtgcaactactcaagtggcaacaggggctacatcaagcagcacctgcgtgtccatcggcagaggcagccttaccagtgtcctatctgtgagcacatagctgagaacagcaaagacttggaaagccacatgatcaaccactgtaaaaccagaatacaccaatgtaagcagtgcaaagaatctttccattacaaggtgagcggcggtctcaagggttggcagggactttatagctgtagtccagccaccccagtttgtttatcaatgaaaatgagttccagagaagtttggtaatttgcccaaaggtacccccaacaaattaccattgaagataggtctggaagctaagtctccatggttgctgtccagagctttcccttagtctgatgccgtgccctttgcagcctcagcgcctgttgctttcATGTTTTCCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGAAGCACGGGTTTTCACTTAGAAGGGAACATAGCGTAGACTGACAGGCACGTGTCTGACGGCAAAGGCTAACTGTGCATCTGCACACAGACCAGTATTCCCTGCACTCATAGCAGCTGCTACACAACCAGCAAACAGTGGTCTCTCACTGCAGAAGGGAAGCAGCCTGTGAGACCTGCTAGGAGGCAGCACAAGAGAAATGATACCATATTTGCTATTGCTGGACACTTTGAGGGACTTTGTTAGGAACCAACTCCAAGACCTCACACCAGCTAGGCAAATGCTTGCCTACCTGTGAAGTGCATTCCCAGCCCTTGTTTTTGTGAGAGTCCTCCTGAATGTCCCAGGCAGAA

A 2580 bp deletion beginning at Chromosome 7 position 35,793,294 bp and ending after 35,795,873 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45148 TGT CAT TGC TAT TGG GGA CA Wild type Forward A
45149 AGA TGA AGA CGG TGG GTC AG Wild type Reverse A
45150 Fluorophore-1 TCC ACA ATG GGC CAT CTG Quencher-1 WT Probe
46376 TTG CAA GGG ATT CAT GTT TTC Mutant Forward A
46377 TAG CCT TTG CCG TCA GAC AC Mutant Reverse A
47215 Fluorophore-2 CAC GGG TTT TCA CTT AGA AGG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45148 0.40 uM
45149 0.40 uM
46376 0.40 uM
46377 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.