Protocol 37929: Probe Assay - Cdc37<em1(IMPC)J>-Altnernate 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

 

>chr9:21140676-21140794 119bp GATGCTGCAAGACGCCATC CTTGGACTCACAACCCTGCT

Mutant=  116 bp

Wild Type = 119 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

TAGTTAAACCTAGTCTTCAAAAAAAACATAGACATGTGGCAGCAGGGCACCAGCGGATGGGTAGCACGTGGCTGTGTGCTCTGTAGTCAGGGCCCAGGGTGCGTCCCTGACCCAGGCCTGTCTGGTCCCCTGTGGTAGCAGGgtaggagttctttctatgactggctgctgattgtccctgcctgcaggcccgggtggaacgcatggagcagtttcagaaggagaaagaggaactggaccggggctgccgggagtgcaagcgcaaggtagccgagtgccagcgcaagctgaaggagctggaagtggctgagagcgatggccaggtggagcttgagcggctgagagctgaggcacagcaactgcgcaaggaggagcggagctgggagcagaagctggaagacatgcgcaaaaaggagaagaacatgccctggaatgtggacacgctcagcaaagatggctttagcaaggtgggacactggcaagagtgctatggagtgcggtcaggctagctgggggtggttcggtggtggaggctagacatgcctgtaaagaccccaggtccacccctgtgtccccatcccagtaaactagaaaaagaattctgacaatgagagccaggcaaagccaccgggtggggtgcgaggacaggggagggacagacagacagacagtttgagcttgggattggctagtggaggccaacgaagcccatccataccctctgatctgcccgaaggcccgcggccatgcctccgccaccttgatgcacttagcgacgccaccgcctccgatgtggacaggatgggcgcggcggccacggcccccaaaccccaggcgcacccctgccttgccccagcccacttgccctctcaccctgtcacagagcatggtcaataccaagcctgagaaggcagaggaggactcagaggaggcaagggagcagaaacacaagaccttcgttgaaaaatatgagaaacagatcaagcatttcggtaagttcctgcacaaaggctaggttaggagtcaaggccagcaaaaggcttccaatggcctctgcgacatcttcctaacatcctctcctgcaggcatgctccaccgctgggatgacagccagaaatacctgtcggacaacgtccacctggtgtgtgaggaaacggccaactacctggttatctggtgcattgacctggaggtagaggaggtaagtgtgcccttcccagctgcggggactcccagacactgcgcctgctcacgccccgcccacctctttgtcctggcagaaatgtgcactgatggagcaggtagctcaccagaccatggtgatgcagtttattctggagctggccaagagtctgaaggtcgacccccgagcctgcttccggcagtttttcaccaagatcaaggtaatgagctcctgtgtaccaccaggaaatccacagtgcagtgcgcgatgtcagatatttattagaggggcttgaggtgccacggggatggccgtacatatgtatacttgtgtgtgtgtgtatacatacatacatacatacatacatacatatatatatgtatgtatgtatatgaaacgtgtgtggatgtggatgtggacccatatgattaagagataactttgtgtggttggttctccacctttctgtgggtccctgtggtaaaacaagctgttaggcttacatggcaaagcactttcacctactgaacacctagctgaccctatttgaggatttctttctattcatttactttaaatattaaataatttatatgtaatgtaaatatattgcataaacattatataagtaaatattaattttttttgttgctgtgtgtgtgcctgagtgagtttatgtgcatcattcgcatgcaagtgtcttcagaggctagaagagggcgttgggtccctggaggtggagttgctagggtttgcaagctgtcatgtgggtaatgggtaccaaacttggttcctctgaaagactggggaacctgagagcagggcagggtagaacaatccagaactgaggtttctgtaagaccttgctggaggacacctgaagatgtgataccaagctggtgctgttgagccgagccaggcggctggccattagcaggtggtctcacaagctagtaggatgggcgctgaatgcaggagacaaaggccaaatcttgaggtagccatggtggagttcaggaaaagcttcagggagccttcacctccctaaacatctggttatgaattgcagtgagcagtacccaccctcttccacagaccgctgaccaccagtacatggagggcttcaagtatgaactggaagcctttaaggagcgagtgcggggccgcgccaagctgcgaatagagaaggccatgaaggaatatgaagaggaggagcgcaagaagaggctaggccctggtggcctggaccccgtggaggtctacgaatccctgcctgaggtgtgactccccagggcccaggagggaggagggtgatgtatacccattgctgctccctgcccccgcccctctgctcccacccatcccaagcccaggccctcaccctggcccctctgtactcaggagctgcagaagtgctttgatgtgaaggatgtacagatgctgcaagacgccatcagcaaaatggatcccactgtgagtccctagtccctgtggcagatggcagatggccagggGGTGCAACCCTGTCATCCAAGAGCAGGGTTGTGAGTCCAAGGCCA

 

A 2,555 bp deletion beginning at Chromosome 9 position 21,140,717 bp and ending after 21,143,271 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49594 CTT GGA CTC ACA ACC CTG CT Common A
52542 GAT GCT GCA AGA CGC CAT C Wild type Forward A
52543 CGT GGC TGT GTG CTC TGT AG Mutant Forward A
52544 Fluorophore-1 CCT AGT CCC TGT GGC AGA TGG Quencher-1 WT Probe
52545 Fluorophore-2 CTG GTC CCC TGT GGT GCA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49594 0.40 uM
52542 0.40 uM
52543 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.