Protocol 28042: Standard PCR Assay - Puromycin
Version 3.2


This assay does not work well without the use of a Hotstart (We are using Taq Start Antibody mixed 1:1 with Taq polymerase).
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

Transgene = 166 bp
Internal positive control = 324 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR1241 ATG ACC GAG TAC AAG CCC AC Mutant Forward A Puro F
oIMR1242 GCG TGA GGA AGA GTT CTT GC Mutant Reverse A Puro R
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT Internal Positive Control Forward A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C Internal Positive Control Reverse A

Reaction A

Component Final Concentration
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR1241 0.50 uM
oIMR1242 0.50 uM
oIMR7338 0.50 uM
oIMR7339 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul


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
024906 129-Dp(16Tiam1-Il10rb)8Yey/J
014168 B6;129-Pot1atm1.1Tdl/J
002355 C57BL/6J-Tg(pPWL88puro)2Ems/J
003208 STOCK Dnmt1tm3Jae Hprtb-m3 Tg(pPWL512hyg)1Ems/J
018121 STOCK Gt(ROSA)26Sortm1(birA)Mejr Gata4tm3.1Wtp/J
028980 STOCK Ppm1dtm1Lad/J
6 strains use this protocol