Protocol 38433: Probe Assay - Cdh20<em1(IMPC)J>-Alternate2
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

>chr1:104940912+104941051 140bp CTGAAGGGGAAAGAACATTC CTGTCCATGTCTGAATGAAGC

Mut= 150 bp

Wt= 140 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

ACAATAATTTTAAAATATTAAGATCATACTGTGTTTGAATACCATAAGAATATGACATAGGAAAACTTCACATATTATTCTTTATCCATTGGTTCTTTACAGACAGACATCCTAAACACTGAAGGGGAAAGAACATTCTTATGTAACGGGAGATCTTAAGTCTGTTCCAAAGTGAAAATCAACTTATTTCCAatacctgtgcatatcaataatgacatttcagtgcttttgtccccagcttcattcagacatggacagaggagatggatccatcaaatacatcctttcaggagaaggagcaggcatcgtgtttaccattgatgacaccactggagacatccatgccattcagaggctcgaccgagaagaaagagcccaatatactctaagggctcaagccctagacaggagaacaggccggccaatggagccagaatcagagttcattatcaaaatccaagatatcaatgacaacgagcccaagttcctggatggaccttacatcgccactgtgccagaaatgtcaccagtaggtaagatggtggtgagctcatttttacaaatagtagatgcattgtaggagatgctactttgtacacatagaggagaaaaggtacagaaatcataaaagtgaaactaaaatgactaccaactccttcaataggtagcttcctgaagctatggagatatcagaagcagccacattaagtaataattttccaatgcctgttagttctgagttcttaagattcaaagacatgaacaatggctgatagattatattgtattagattagattagattagattagattagattagattgttaaggtaaatattggtattaccaataaacaaggaccacataggcagtgttacttttactggctactaagaatttaacacagctgggcgtggtggcgcatgcctttaatcccagcacttgggaggcagaggcaggtgaatttctgagtttgagggcagcctggtctacagagtgagttccaggacagccagggctacacagagaaaccctgccttgaaaaataaaataaaataaaataaaataaaataaaataaaataaaataaaataaaatttaaaaaaaagaaagaaagaatttaacacatatgactatcatccctattgagtaacaaaatgggagtcatggaatcgtgatcaagaaagggaactctcccagagaagcaagcaacatctgtaaAATGTTTTAAGGTTGACTATGCAAGTACTATACCAGCTGTTTTAAACTGGGCTTCATTAGGTTTCTCCCACTTCTCTCCCTCAGGTACATCTGTTATCCAAGTGACAGCCACAGATGCTGATGACCCAACCTATGGCAACAGTGCCCGGGTAGTGTACAGCATTCTCCAGGGCCAGCCATATTTTTCTGTGGACTCCAAAAC

This mutation is a 1028 bp deletion beginning at Chromosome 1 position 104,940,986 bp and ending after 104,942,013 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53808 CTG AAG GGG AAA GAA CAT TC Common A
53809 CTG TCC ATG TCT GAA TGA AGC Wild type Reverse A
53810 GAG AAG TGG GAG AAA CCT AAT GA Mutant Reverse A
53811 Fluorophore-1 ATT TCA GTG CTT TTG TCC CCA Quencher-1 WT Probe
53812 Fluorophore-2 ACT ATA CCA GCT GTT TTA AAC TGG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53808 0.40 uM
53809 0.40 uM
53810 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.