Protocol 35047: Probe Assay - C2cd2l<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

>chr9:44317095-44317186 92bp TAGAAGTGGGGAAGGGCAGA CAGCAGAGAGCTGGCAATG

Mutant=  96 bp

Wild Type = 92 bp

Sequence

TCTAGGGAGAGAAGAGTTTTTGTCTTTGTTTTGAGACAGGATTTTACTGTGTAGGTCTGCCTGTCCTGGAACTCACTCTGTAGACCAGGAAATCTGCCTGCCTCTGCCTCCAGAGTACTGGGATTAAAGGCGTGCGCCACCACCACCTGGCTGAGATTTTTGCTTTTGTCTAATTCATTGAGAAGCTTAGGGCCCCCAAAAGGACATCATCCCAAGGATGATAGAAGTGGGGAAGGGCAGAGCCAGGTGGGTGTCCTCcgcagtgcctgacactgttccctgtttcaggtgctgcattgccagctctctgctgaggaggtgcgcttccccatctctgtgacccagcagtcccccgctgccgtctccatggagacctaccacgtcactctgacactgccaccaacacaggtggagggaaatgggggaaactgagttgggcagagggcagttgagttggcccagcattttctccctgcttcctctctaccgttggattcgggaaggaagggtccgagtagttagcagggtggggggatggggtcctgaagaatggggtagatcacagtaatccttccgttcttgtctctagttggaagtcagcctggaggaaatccctgatgaggggctcctggtgtcctgggccttcactgaccgcccagaactcagcctaaaggtgcttcccaagttgcagactagggaggtaagaagggggcagcagaggagagactgaactggcgcaatggagcagggcggaggctccacctcttctcctctctgcagagagatgaggaacaaccagagctctcaacagttgaggaactgatcaaggacgctatagtcagcactcagcccgccatgatggtcaacctcagggcctgctctgccccaggaggcctggtgagtggacacccaaagggaaggtttgcgtgtatcccacctccaggttactatatcgggattatttctctttttactcccttttgctcttaggtacccagtgagaagccacccacgatgtcccaggcccagccatccatccccagacctacccgattattcttacggcagcttcgagcatctcacctgggaagtgagctaggaggtgagaattccgctgcccaggacatagcttctctattccgcggacttggtttcttccgtctgagcatcctctaagctctgcttcctgacaggtactgaggaactgtgctgtgccgctgagcttgacaaccccatgcaacaaaagtggaccaaacccatgagggcgggccccgaggtggaatggaccgaggacctagctctgtaagtcacactctgctcctcccctctgtccccatgggcagtcaggatgaagaaacttttccaggctagccctggcatgaggaagccagccttgttttcagtagcctgtagtgtggacctccactgtatgccaggccctggggacacagactaggagtagatacagccctacctctgggtggatcacagacccgaagcagagacacatagggaagaaaggacaaagttgccagttgagattggtgcacagcatgcCGTGGAGGCGCTTCAGTAGGGGAGATGACGAATTCTCAAAACGGACTAAAGAACTGATGAAATGTCATGGAAGAAGTCTATTTAAACCACGCCTTCAAGCAAGGGTAGCACCGCAACAGAATGTGAAAGTCTTCTT

A 1299 bp deletion beginning at Chromosome 9 position 44,315,851 bp and ending after 44,317,149 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45127 TAG AAG TGG GGA AGG GCA GA Common A
45128 CAG CAG AGA GCT GGC AAT G Wild type Reverse A
45129 CAT CAG TTC TTT AGT CCG TTT TGA G Mutant Reverse A
45130 Fluorophore-1 AGT GCC TGA CAC TGT TCC CT Quencher-1 WT Probe
45131 Fluorophore-2 TGG AGG CGC TTC AGT AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
45127 0.50 uM
45128 0.50 uM
45129 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.