Stock No: 005703
Protocol 24532: Standard PCR Assay - Tg(ACTFLPe)
Version 4.2

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

This assay can be used to detect FLPe.

This assay cannot be used to detect FLPo.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
17772 TGC CGG TCC TAT TTA CTC GT Transgene Forward A
17773 TAC TTC TTT AGC GCA AGG GGT AG Transgene Reverse A
oIMR8744 CAA ATG TTG CTT GTC TGG TG Internal Positive Control Forward A
oIMR8745 GTC AGT CGA GTG CAC AGT TT Internal Positive Control Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
17772 0.50 uM
17773 0.50 uM
oIMR8744 0.50 uM
oIMR8745 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
021072 B6(Cg)-Slc6a8tm1.2Clar/J
021828 B6(SJL)-Lrrk2tm3.1Mjff/J
025930 B6(SJL)-Pik3cbtm1.1Jdo/J
018445 B6.129-Mre11atm1.1Rchd/J
022364 B6.129P2(SJL)-Ntrk3tm1Ddg/J
018540 B6.129S6(Cg)-Bpnt1tm2.1Yrk/J
012839 B6.129X1(Cg)-Tnfrsf4tm2(cre)Nik/J
021191 B6.Cg-Pvalbtm2.1(flpe)Hze/J
013717 B6.Cg-Slc20a1tm1.2Cmg/J
012627 B6.Cg-Stard4tm1.1Bres/J
012631 B6.Cg-Tyrc-2J Sharpintm1.1Sun/J
005703 B6.Cg-Tg(ACTFLPe)9205Dym/J
012653 B6;129-Pax7tm1.1Fan/J
014168 B6;129-Pot1atm1.1Tdl/J
012623 B6;129S4-Carftm1.1Aew/J
016168 B6;129S4-Fbxl3Gt(FHCRC-GT-S13-12G1)Sor/JtJ
018353 B6;129S4-Ror1tm1.1Meg/J
034712 B6J.Cg-Cyp27a1tm1.1Dpm/J
034731 B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J
022365 C57BL/6-Il15ratm2.1Ama/J
035185 STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ
023199 STOCK Brsk2tm2.1Jrs/J
010702 STOCK Gad2tm1(cre/ERT2)Zjh/J
022972 STOCK Pros1tm1Grl/J
24 strains use this protocol