Protocol 20000: Probe Assay - Drd2<tm1.1Mrub> PROBE-Alternate 1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Muant=  165 bp

Wild Type = 77 bp

Sequence

Wt Sequence: TGGACCAGCTGGTCAGAGAAAATGTATGTCTTCTCTACtcacattgcaaaaaaaaGGTCCAAGCTGGAATTctgccttgccCTGGAATTGTTTCAGAAGATTATACTAGAAGCAGGGCTAAATGGTGAGAATCCCTAAGGAGGGGACAATGCCTGTGCTCCAAGGCCTGAAACTCCTGTTGCTCT

 

Mutant Sequence:

GGTCAGAGAAAATGTATGTCTTCTCTACTCACATTGCAAAAAAAAGGTCCAAGCTGGAATTaattcgatgatccggaacccttaatataacttcgtataatgtatgctatacgaagttattaggtccctcgacctgcagcccaagctgatcctctaattCTGGAATTGTTTCAGAAGATTATACTAGAAGCAGGGCTAAATGGTGAGAATCCCTAAGGAGGGGACAATGCCTGTGCT

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
29634 AAA AAG GTC CAA GCT GGA AT Forward A
29635 CAC CAT TTA GCC CTG CTT CT Reverse A
29636 Fluorophore-1 TGC CTT GCC CTG GAA TT Quencher-1 WT Probe
29637 Fluorophore-2 CCT GCA GCC CAA GCT GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
29634 0.40 uM
29635 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.