Stock No: 033185
Protocol 35599: Probe Assay - Adcy5<em4(del101)Lutzy>
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=  75 bp

Wild Type = 83 bp 

>chr16:35279300+35279382 83bp GAACCCAGAGGATGAAGTGG GGACGTGTTCAGATCGCAGT

Sequence

Wt Sequence:
CCAGGAAAGTGCGAACCCAGAGGATGAAGTGGACGAGTTTCTGGgtcgggccatcgatgccaggagcatcgacagactgcgatctgaacacgtccgcaagttcctc  
 
Mutant Sequence:
ttccagGAATGCCCAGGAAAGTGCGAACCCAGAGGATGAAGTGGACGAGTTTCTGgcATCAGGAGAGAGGGCCCCTGGCAGAAGCAAGGCAGGGCATAGAGGGCAGCCCACAGGG
 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46480 Fluorophore-1 CAT CGA TGC CAG GAG CAT Quencher-1 WT Probe
46482 GAA CCC AGA GGA TGA AGT GG Common A
46483 GGA CGT GTT CAG ATC GCA GT Wild type Reverse A
46486 CTA TGC CCT GCC TTG CTT CT Mutant Reverse A
46487 Fluorophore-2 CGA GTT TCT GGC ATC AGG AG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46482 0.40 uM
46483 0.40 uM
46486 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.