Protocol 37670: Probe Assay - Zbtb12<em1(IMPC)J> Alternate 3
Version 4.0

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

>chr17:34897088+34897222 135bp CTGGGGAAGTGGGAAAGTAGA CCCTCCGCTACTCACTGTTC

Mut = 105 bp

Wt = 135 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

CCACCCCTTTATCCATTCTCCCGTGGGCGCGCGGGCTGCGGGACTGGGGGGTGTGGACGCCGCTGCCCCGCCCCCGGGAACCACGCCTCCCTCCTCGGAGGGACCGCGCCTCCTTggaattggggaaactgagggaggcgcgccaagactagggaggtagggatgaggggtctggagccttggcctccgacagccagagacctttgtctctgactcgtttccttcccacccagggcagtccactcccgccacaatggcctctggggtggaagtcctgcgcttccagctgcctggccacgaggccgcaacgttgcggaacatgaaccagctccgtgcagaggagcggttctgtgatgtgaccatcgtggccgacagcctcaaattccgtggccacaaggtcatcctggccgcctgctcaccgttcctccgggaccagttcttgctgaaccccagttctgagctgcaggtctcactgatgcacagcgcgcgtattgtggcggacttgctcctctcttgctacacaggcgccctggagttcgcagtcagggacatcgtgaactacctgacagcagcctcctacctgcaaatggagcatgtagtggagaaatgccggaacgcccttagccagttcattgagcccaaaataggccttaaagaagatggggtcagcgaggctagcctcctaagcagtgtcagtgccaccaagtccctcctccctccagccaggaccccaaagccagctccaaaacccccaccaccacctcctctaccccctccgctcctgcgaccggtgaagctggagtttccgctggatgaggacctagagctgaaggcggaagaggaggatgaggatgaagatgaagatgtttctgacatctgcatcgtcaaagtggagtcggctctagaagtggcacaccggctcaaaccccctggaagcctagccgggggtctgggtatcggggcctctgtgagcagccacctgggagagctggcccagagtagtgtggcccccaacactgttaccccaccgcaaggggtggtgaaggcatgctacagcctgtcagaggacgcggaaggggaaggcctgttgttgatcccaggaggccgggccagtgtgggggccacctcgggcctagtggaagcagcagcggtggccatggctgcccggggggcggggggcagtctgggggcagggggtagccggggacctctgcctgggggcttctcaagtggaaaccccttaaagaacatcaaatgcaccaaatgccctgaagtgttccagggtgtggagaagctggtcttccacatgcgtgcgcaacacttcatcttcatgtgcccacgctgcggcaagcagttcaaccacagcagcaacctcaaccgccacatgaacgtccaccgtggtgtcaagtcccattcatgtggcatctgcggcaagtgctttactcagaagtccacactgcacgatcacctcaacctgcactcaggagcccggccctaccggtgctcctactgtgacgtgcgctttgcccacaagcccgccattaggcggcacctcaaggagcagcatggcaaaaccacagctgagaacgtgctggaagctggtgtggctgagatcaatgtcctcatccgctgaaaggccaagaggctgggggcggtcctgggcccggcagaattggggaggggcggggtttgagggcaggcaaacattttgagggaggaagactggagagagagagagaccatgcctcaaagagcagaagaaaaagatggagccggagacagggttgataaggctgagggaatgtgggatcccaaagaaagatttcttttttgaagaggtgcataaacgatgtggaggaagagagggaaaaaaaaaaaaaggcaaagttgaaagttttggagaacagctctttcctggctaaagctggctgggggggggggggattctagcgtttcttgagatggggtgggggtgggaattgtagggggattgccaggaagcagcttacttgcccctggttagaatagatgacagggagaagggagcggagggcgacacatgtatgaagtaccagaaggaagggaggagatttttgcatgctggggaagtgggaaagtagatacaaggctcaccggaaatgaaatgatatgtgccaggtaagagaacaCGGTGGGCAACCTGGGTAGGTAATGAGACATTAGGCTACAGTAGCGAGAACAGTGAGTAGCGGAGGGGTGGGGGGGCAGGGCACTTAGAAAAAAAGGACACAAGGAGGGCTGTTGGGGGGAGGGAGAGGAGTAGAGACATAC

This mutation is a 2053 bp deletion beginning at Chromosome 17 position 34,895,103 bp and ending after 34,897,155 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51306 CTG GGG AAG TGG GAA AGT AGA Wild type Forward A
51307 Fluorophore-1 CAA GGC TCA CCG GAA ATG AAA T Quencher-1 WT Probe
51759 CCC TCC GCT ACT CAC TGT TC Common A
51760 GAA CCA CGC CTC CCT CCT Mutant Forward A
51929 Fluorophore-2 AGG GAC CGC GCC TCC TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51306 0.40 uM
51759 0.40 uM
51760 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.