Genotyping Protocols Database
Stock No: 004669
Protocol 29763: Standard PCR Assay - Itgb3<tm1Hyn>alternate 2
Version 1.2

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 = ~210 bp
Heterozygote = ~210 bp and 139 bp
Wild type = 139 bp

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
22419 ATG AAA CCA GGA GGA AAG CA Common A
22420 GCC TGG GTG CTC GGT TTA Wild type Reverse A
oIMR7415 GCC AGA GGC CAC TTG TGT AG Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
22419 0.50 uM
22420 0.50 uM
oIMR7415 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
004669 B6;129S2-Itgb3tm1Hyn/J
008819 B6.129S2-Itgb3tm1Hyn/JSemJ
2 strains use this protocol