Protocol 23077: Standard PCR Assay - Generic iCre
Version 1.3

Notes

Transgene = 236 bp
Internal positive control = 324 bp

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

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

This assay does not work well without the use of a Hotstart Taq polymerase.

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
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
oIMR9266 AGA TGC CAG GAC ATC AGG AAC CTG Transgene Forward A iCre F
oIMR9267 ATC AGC CAC ACC AGA CAC AGA GAT C Transgene Reverse A iCre R

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR7338 0.50 uM
oIMR7339 0.50 uM
oIMR9266 0.50 uM
oIMR9267 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
009587 B6(129S4)-Et(icre)1402Rdav/J
009588 B6(129S4)-Et(icre)1470Rdav/J
009589 B6(129S4)-Et(icre)1555Rdav/J
009586 B6(129S4)-Et(icre)754Rdav/J
010696 B6(129S4)-Et(icre/ERT2)10596Rdav/J
010697 B6(129S4)-Et(icre/ERT2)10727Rdav/J
012689 B6(129S4)-Et(icre/ERT2)14163Rdav/J
012690 B6(129S4)-Et(icre/ERT2)14208Rdav/J
012694 B6(129S4)-Et(icre/ERT2)14915Rdav/J
008610 B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J
008520 B6.Cg-Tg(CD2-icre)4Kio/J
030663 B6;129S-Gcgtm1.1(icre)Gkg/J
018968 B6N.Cg-Commd10Tg(Vav1-icre)A2Kio/J
030676 C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J
019098 FVB-Tg(Csf1r-cre/Esr1*)1Jwp/J
012691 STOCK Et(icre/ERT2)14374Rdav/J
012692 STOCK Et(icre/ERT2)14602Rdav/J
012693 STOCK Et(icre/ERT2)14624Rdav/J
18 strains use this protocol