Genotyping Protocols Database
Stock No: 005582
Protocol 27927: Standard PCR Assay - Cx3cr1<tm1Litt>alternate1
Version 3.2

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 = ~500 bp
Heterozygote = 410 bp and ~500 bp
Wild type = 410 bp

Mutant      Tm = 83    Area = 26    Tm = 89   Area = 36   (WT  Tm = 81  Area = 7     Tm = 84   Area = 15)

Wild type  Tm = 89   Area = 45

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
14276 GTC TTC ACG TTC GGT CTG GT Wild type Forward A
14277 CCC AGA CAC TCG TTG TCC TT Common A
14278 CTC CCC CTG AAC CTG AAA C Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
14276 0.50 uM
14277 0.50 uM
14278 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 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

Stock Number Strain Name
005582 B6.129P2(Cg)-Cx3cr1tm1Litt/J
008451 B6.Cg-Ptprca Cx3cr1tm1Litt/LittJ
2 strains use this protocol