Protocol 37970: Probe Assay - Zfp286<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

>chr11:62779690-62779821 132bp TTCGTCAGCTCTTATTCAACACC GGCTGCTCTTCAGTGTGAGT

Mut= 140 bp

Wt= 132 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

TGCGTTATATTTCTGATCCCCTTTCTCCCCTCCGATTCTCCTGGATAAAGCTATTCGTCTTCATATTCATCCCCCTCGATTTCCTTCTTATTTTACAAACCATCCAGTAAGAGCGTCAGATCTCCTAACTCCACAAAGGCATGCTTCCATCTTGATTCATCTTCACCTGTTATAGGACTGTGTTACTGTTGAGCACAGAAAACAAAGAACATGTGCATTTGCAATGTCTTTTCAGACATGGAAACcagacctgagagcaaggagtcaacttcagtgctagacttttccaaagaagagtcaaacagactgacagaaaatactgcttctgactccgacattgaaactgctcttgaatgtgagcgttggctagaacatcgtcaaagaaatcgggagaggtacccaagacaaatgtctgctcacaggaactccctccctgcgcaagaagatggtgaacgtgatgtatattggaaaagcttcagtcagaaacctctccttacaactgatgacagagaccccaaaggatcctgggacgttcacacgcttgggaaaagactgaagcagaaatcaaagttaatgacaaagcagagaccttataaagagaaaaaacctcataagtgtgatgagtgtggcgaactcttcacttaccgttcggtacttatccgacaccagagagttcacactggagagaaaccttatacctgcagtgagtgcgggaagtccttcagccacagagccaatctgactaagcaccagagaacccacactaggattctcttcgagtgtggcgagtgcaagaaaaccttcatagaaagttcatcccttgaggtacatcagagaatccacatcggggagagaccttacgaatgcgccgagtgtgggaaaggcttcaaccgaagtacacaccttgcccagcatcagctgattcatactggggtgaagccttatgaatgtaacgaatgtgacaaagctttcattcactcatcagctctcattaagcatcaaagaactcatactggagagaagccctataaatgtcaagactgtgggaaagccttcagccactgctcatctcttacgaagcaccagagggttcacacgggggaaaggccatatgagtgcagtgagtgtgggaagactttcagtcagagcacgcaccttgttcagcaccagagaatccacactggagaaaagccctacgaatgtaacgaatgtgggaaaacattcagccggagctcaaattttgcaaagcatcaaagaattcacatgggaaagaaaccctataaatgtggagaatgtggcaaagccttcgttcattcgtcagctcttattcaacaccagagaactcacactggggagaagcctttcaggtgtaccgagtgtgggaaaagcttcaagagcagcccctccctcatcCGACATCAAAGAACTCACACTGAAGAGCAGCCCTGAGAGGTGCTGAGTGTTAGGTGAAGGTTGGCTTTATTGCTGCGTTTCATATTTGATTGTTGAAATAGAGAGACCATAATATGACCCTGGGGTGTGTCTGTGTGTGTATGATTTCACTTGCCCTGTGTATAACTCTGAACAGCAGAAAGGAGATCCCTTCTACCCATTGGCTTGATAATAGTGAAGCCCTCT

This mutation is a 1168 bp deletion beginning at Chromosome 11 position 62,779,722 bp and ending after 62,780,889 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52659 TTC GTC AGC TCT TAT TCA ACA CC Wild type Forward A
52660 GGC TGC TCT TCA GTG TGA GT Common A
52661 GGC ATG CTT CCA TCT TGA TTC Mutant Forward A
52662 Fluorophore-1 AGC CTT TCA GGT GTA CCG AGT GT Quencher-1 WT Probe
52663 Fluorophore-2 CTT TTC AGA CAT GGA AAC CGA CAT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52659 0.40 uM
52660 0.40 uM
52661 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.