Protocol 32775: Probe Assay - Cherp<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

>chr8:72471121-72471217 97bp TGGGACAGTGAGATCAAGCA AGGTTGCCCTCCAGAGAGAG

Mut= 98 bp

Wt= 97 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (CT insertion):

 

ATTAGGATCCAGTGGCTTGGGACAGACACACCTTGGGAGAGGTGCCACATCTCCAGCCCTGGTCATAGCTTGCAGTGCATAGTAGCTTGGTCAGGCCTGCATGAGAAGGGAAGATGGAAGGGTCAGCCTGTCTTGGGACAGTGAGATCAAGCAC^aacagggaagcttgggcaggatggccgtgtgcggtggacagtggatccatcaggtcctctctctggagggcaacctgatggatcaccaaaaggaacctggggcaccccctgagctcacaaggcccatctctttcctgctagtaatctgcaagcagcaggccccagagctggagcccacctcagccatgccccctctgccacaacctccgctggctcccacagcgtccctcactccggcccagggcaccccatcaatggacgagctcatccaacagagccagtggagtctccagcagcaggaacagcacctgctggccctgcgccaggtatgcctgcatgtactgtcagtgaccctggagtcaccttccagtaggtctgtgggtcatccacaggcatcgctcagtggacttgtacccattgtggatgccaacacatggcggcttaggtacctaccattcttaatgagtagggaggggcattattggccccatcttttaggttggttctgttgccaaattctatagg^GATGAGCTACCTGAAATGAAAGGAGAGAAGACATACAGACCTGAGTGCCAGCCCCACTCAGGGTTTCCTTGCTTACCACCATCACATATCATGCATGGATGTTTACCATCAGTCATAGTCTGGACAGTTAGGTCTTAGACAGCAGAACCAGCCTCTACTCTTGGTGACCTATAC

This mutation is a 525 bp deletion beginning at Chromosome 8 position 72,470,672 bp and ending after 72,471,196 bp (GRCm38/mm10). In addition, there is a 2 bp insertion (CT) at the deletion site.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39949 TGG GAC AGT GAG ATC AAG CA Common A
39950 AGG TTG CCC TCC AGA GAG AG Wild type Reverse A
39951 TAA GCA AGG AAA CCC TGA GTG Mutant Reverse A
39952 Fluorophore-1 ACA GGG AAG CTT GGG CA Quencher-1 WT Probe
39953 Fluorophore-2 CCT GAT GAG CTA CCT GAA ATG AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39949 0.40 uM
39950 0.40 uM
39951 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.