Stock No: 033183
Protocol 35597: End Point Analysis Assay - Adcy5<em#(A727T)Lutzy>
Version 1.0

Notes

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 = ACA

Wild type = GCC 

>chr16:35279312+35279390 79bp TGAAGTGGACGAGTTTCTGG GAACTTGCGGACGTGTTCAG

Sequence

gGAATGCCCAGGAAAGTGCGAACCCAGAGGATGAAGTGGACGAGTTTCTGGGTCGGGCCATCGAT(g/a)c(c/a)AGGAGCATCGACAGACTGCGATCTGAACACGTCCGCAAGTTCCTCCTGACCTTCAGGGAGCCCGACTTA
GAGAAGAAGgtatggcatcaggagagagggcc

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46478 TGA AGT GGA CGA GTT TCT GG Forward A
46479 GAA CTT GCG GAC GTG TTC AG Reverse A
46480 Fluorophore-1 CAT CGA TGC CAG GAG CAT Quencher-1 WT Probe
46481 Fluorophore-2 CCA TCG ATA CAA GGA GCA TCG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46478 0.40 uM
46479 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 4.0 -- 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.