Protocol 36395: Probe Assay - Zbtb37<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

>chr1:161031484-161031574 91bp TCAGTGAATCAGAATAGCATCG AGACCCCTGACAATCAGCTC

Mut= 91 bp

Wt= 91 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AATACCTTGTACCAGGCTCGGCAGCTTACAGTAATCCTAGCACTTAGGAGGTAGGATCAGGAGTTTGAGGCCAGCCTTATTACTAACAAAACACCTGCTGGCAATTCTGTTTCTACATCCAAACATAAGGAAGATCTCTCTTTCTATATGAAGAATTCTTTTTTTCTCCCTGAGACAAGATTTCACTTATTTAGCCCATACTCATGATTCTCCTGCCTCTTCGGTTTTCCAAGCGCTAAAATTACGTATGTGTAGCTGGCACTCATGGAAAGAttttccatttgtagggatgtttgaactaaaaagcacaaagggagtcaaactagtactaagactgtgctttgctctttcagcagggttgaatgtctgcctgtgctttcaggcatgaccatggagaaaagtgggaacatacaattggagatccccgacttcagcaactctgttctgagccatctcaaccagctacgaatgcaaggccggctctgtgatattgtggtcaatgtgcaaggacaagcttttcgggctcacaaagtagtactggctgccagctccccttatttccgggatcacatgtccttgaatgaaatgagtactgtgtccatttctgtcatcaagaaccctactgtttttgagcaacttctttctttctgttacacagggcggatatgcctgcaactagcagatatcatcagctacctaacagctgctagttttctgcagatgcagcatattatagacaaatgtacgcagatcctagagggcattcacttcaaaattaatgtggctgaggttgaagcagaattaagtcaaacaaggacaaagcatcaagagagacctccagagtctcatagggctacaccaaatctaaaccgctcacttagtccacggcataatgcctcaaagggaaactggagagggcaggtcagtgccgtgctggatatccgagagctcagtcccccggaggagcccaccagtcctcagataattgaacagagttctgatgcagaaggccgggagcccattcttcgaatcaacagagcaggacagtggtatgtggaaacaggactagcagaccagggggcccggagtggtgacgaagttagggttcttggagcagtgcacatcaaaactgaaaatctagaagagtggcttgggccagagaatcagccttctggagaagatgggagtagtgcagaggaagtcacagccatggtgattgacaccacaggccatggttctataggacaggaaagttatactttagggtcttcaggagccaaggtagctcggccaaccagcagtgaagttgacaggtaagttttgtttctccttttaaagctattgtacagacatcattaaataacctttctgtgttacagagggcatctttttttacctgatattcttattcagcttactttgggagactggaagtatgtcagaagacttgcattttcagcacaggaggctcctggatctgaaatattgacgatgttatgagaactgtggtaaactgtggtaaactgtggtaaaattagccacttcagtgaatcagaatagcatcggcagccactctaggatcagtaattgccagggctctcTGCACTAGGCAGGGAGCTGATTGTCAGGGGTCTGGTAACATTGCTGTTTCTCACACAGGTAGGTAACCTTGCGGAGTCTGACTTGCCTTATAGCACTGTGTATTTCTCTTTGCCCTTACCGACAGAGACAATTTGGTACCATCAATAATTTTCAAATTAGAGTAATTACACTACAGTAATTCTAGAGAAAAGCTTTTGAATGTTTTCCAAGTGTCACCTGGTAATGTTTCCTTTGAAGCAGCTACTACCCCAGGCCTGTTTCTTCCTAACATACTGAGTCTG

This mutation is a 1328 bp deletion beginning at Chromosome 1 position 161,031,517 bp and ending after 161,032,844 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48635 TCA GTG AAT CAG AAT AGC ATC G Wild type Forward A
48636 AGA CCC CTG ACA ATC AGC TC Common A
48637 CCT CTT CGG TTT TCC AAG C Mutant Forward A
48638 Fluorophore-1 CTA GGA TCA GTA ATT GCC AGG G Quencher-1 WT Probe
48639 Fluorophore-2 CGT ATG TGT AGC TGG CAC TCA T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48635 0.40 uM
48636 0.40 uM
48637 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.