Protocol 28752: Standard PCR Assay - Tg(ACTFLPe)

STOCK Gad2^{tm1(cre/ERT2)Zjh}/J

Stock No: 010702

Version 5.2

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 cannot disquinsh between hemizygotes and homozygotes

 

Transgene- 100bp

Internal Positive Control 324bp

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
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
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
oIMR7338	0.50 uM
oIMR7339	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)-Slc6a8^tm1.2Clar/J
018445	B6.129-Mre11a^tm1.1Rchd/J
022364	B6.129P2(SJL)-Ntrk3^tm1Ddg/J
018540	B6.129S6(Cg)-Bpnt1^tm2.1Yrk/J
034712	B6.Cg-Cyp27a1^tm1.1Dpm/J
021191	B6.Cg-Pvalb^tm2.1(flpe)Hze/J
013717	B6.Cg-Slc20a1^tm1.2Cmg/J
012627	B6.Cg-Stard4^tm1.1Bres/J
005703	B6.Cg-Tg(ACTFLPe)9205Dym/J
036551	B6.FVB(Cg)-Tg(Pmch-cre)1Lowl/J
012653	B6;129-Pax7^tm1.1Fan/J
014168	B6;129-Pot1a^tm1.1Tdl/J
012623	B6;129S4-Carf^tm1.1Aew/J
016168	B6;129S4-Fbxl3^Gt(FHCRC-GT-S13-12G1)Sor/JtJ
018353	B6;129S4-Ror1^tm1.1Meg/J
034731	B6N(129S4)-Srd5a3^tm1c(EUCOMM)Wtsi/J
019100	B6N.Cg-Tg(ACTFLPe)9205Dym/CjDswJ
035185	STOCK Adgrl1^tm2c(EUCOMM)Hmgu/SudJ
010702	STOCK Gad2^tm1(cre/ERT2)Zjh/J
014099	STOCK Tg(Pmch-cre)1Lowl/J
20 strains use this protocol