Protocol 36830: Probe Assay - Zfp74<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

Mutant=  82 bp

Wild Type = 93 bp

>chr7:29937735-29937827 93bp GTGGCATTCCAGGGTACAAC CACACGAGTGACAAAAACAACC

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion): ATTTAGAGTCTGGGTAGGTGTGCTGACTTCACTTAAGGAAGGTTCGGGACAGTAGCATCTTAGGCACTGTGATTACCAGTGTGATGGGCAGGATTGAAGCTTCCCAACATGCTGAATTCACCATGACAACAGGGGAGGCCAAAACCTCCCTAGCACTCTGTGCTCGCTGGGGAAACAGGCAAGGCTGaagtctcccaggtatgcttgtttattgggtgtcatttttctttatagctgaacaaaattctactatatatacataccactggctctgttttctagtaactgagtataaactgactatattgctaatatgacgtttgcttcattttgtttagaaaatgggcagtgctttttgtaatgtcatctttctgaattatatctgaacaaggcttgttatgtttttcctacaccactagaatggataacttcagtttgagtgatgcgtattgttacagggactagtgacgttcaaagacgtggccattgattttacccaagaagaatggaagcaactggatcctactcagcggaacctatataggaatgtgatgctagaaaactacaacaacttaatcacagtaggtaagataatgtaccttttctaaaacagatgcaattgttaatgttgatgagccttgccttgaggtgccttgagatgttgctgtccctgccccagctttcgaaggaaaatctttggtggcattccagggtacaacattctcagaagttagtacacctggctctcccaggcccaactcagaatccagggttgtttttgtcactcgtgtgtatacccaaaccaatgtcatcccctcccccaccccccaggcccgccactcaccaaacctgaagtgattttcaagttggagcaagaggaagagccttgtgtggtggagagagaagtgctgtggagaccctgtccaggttagtgtgtgtgtgtgtgtggggggtgggggggacccagccaggtaaagcgagaggaccagtaagtgactggcctgagatgtttcctaaggtagattcccacagcgtgtagtaacagttatgtgaaactaagatgttctctttaaaaaaaaaaaaaaaattccctgcacttgggaggcagAGGCAGGTGGATTTCTGAGTTTGAGGCCAGCCTGGCCTACAAAGTGAGTTCCAGAACAGCCAGGGCTATGCAGAGAAACCCTGTCTCAGAAAAAAAATTTTTTTTAATTACGTTTTTTTGTGTACACACATGTG

Mutation details

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49756 GTG GCA TTC CAG GGT ACA AC Wild type Forward A
49757 CAC ACG AGT GAC AAA AAC AAC C Wild type Reverse A
49758 CCA AAA CCT CCC TAG CAC TCT Mutant Forward A
49759 AGG CTG GCC TCA AAC TCA G Mutant Reverse A
49760 Fluorophore-1 CAC CTG GCT CTC CCA GGC C Quencher-1 WT Probe
49761 Fluorophore-2 ACA GGC AAG GCT GAG GCA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49756 0.40 uM
49757 0.40 uM
49758 0.40 uM
49759 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.