Protocol 29550: Standard PCR Assay - Generic iCre
Version 4.2

Notes

Melting curve analysis is done using a Roche Light Cycler 480.

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

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 = 236 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
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
012687 B6(129S4)-Tg(SYN1-icre/mRFP1)9934Rdav/J
008610 B6.Cg-Commd10Tg(Vav1-icre)A2Kio/J
023677 B6.Cg-Hprttm332(Ple272-icre/ERT2)Ems/Mmjax
023690 B6.Cg-Hprttm345a(icre/ERT2)Ems/Mmjax
023691 B6.Cg-Hprttm346a(Ple264-icre/ERT2)Ems/Mmjax
023692 B6.Cg-Hprttm347a(Ple155-icre/ERT2)Ems/Mmjax
023693 B6.Cg-Hprttm348a(Ple265-icre/ERT2)Ems/Mmjax
023694 B6.Cg-Hprttm349a(hs671-icre/ERT2)Ems/Mmjax
023695 B6.Cg-Hprttm350a(Ple232-icre/ERT2)Ems/Mmjax
023696 B6.Cg-Hprttm351a(Ple198-icre/ERT2)Ems/Mmjax
023697 B6.Cg-Hprttm352a(Hspa1a-icre/ERT2)Ems/Mmjax
023698 B6.Cg-Hprttm353(Ple274-icre/ERT2)Ems/Mmjax
023699 B6.Cg-Hprttm354(Ple283-icre/ERT2)Ems/Mmjax
023705 B6.Cg-Hprttm360a(Ple253-icre/ERT2)Ems/Mmjax
023706 B6.Cg-Hprttm361a(Ple267-icre/ERT2)Ems/Mmjax
023708 B6.Cg-Hprttm363a(hs1218-icre/ERT2)Ems/Mmjax
023709 B6.Cg-Hprttm364a(CAG-icre/ERT2)Ems/Mmjax
023710 B6.Cg-Hprttm365a(Ple266-icre/ERT2)Ems/Mmjax
023711 B6.Cg-Hprttm366a(Ple67-icre/ERT2)Ems/Mmjax
023712 B6.Cg-Hprttm367a(Ple251-icre/ERT2)Ems/Mmjax
017490 B6.FVB-Tg(Stra8-icre)1Reb/LguJ
029231 B6;129-Bhlha15tm4Skz/J
030663 B6;129S-Gcgtm1.1(icre)Gkg/J
026555 B6;CBA-Tg(Lhx6-icre)1Kess/J
018968 B6N.Cg-Commd10Tg(Vav1-icre)A2Kio/J
030676 C57BL/6N-Tg(Tg-cre/ERT2)1Kero/J
021024 FVB-Tg(Csf1r-icre)1Jwp/J
008694 NOD/ShiLt-Tg(Foxp3-EGFP/cre)1cJbs/J
012691 STOCK Et(icre/ERT2)14374Rdav/J
012692 STOCK Et(icre/ERT2)14602Rdav/J
012693 STOCK Et(icre/ERT2)14624Rdav/J
027798 STOCK Tg(Nkx6-2-icre)1Kess/SshiJ
018147 STOCK Tg(Slc17a8-icre)1Edw/SealJ
008208 STOCK Tg(Stra8-icre)1Reb/J
43 strains use this protocol