Master Protocol Details



Expected Results:	Transgene = 435 bp Internal positive control = 200 bp



Gel Information:	Separated by gel electrophoresis on a 1.5% agarose gel.

Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
oIMR1528	-	GCG CTG ATG ATG TTG TTG AT	-	Transgene
oIMR1529	-	CAT CCC CGA ACC TTT TGA TA	-	Transgene
oIMR8744	-	CAA ATG TTG CTT GTC TGG TG	-	Internal Positive Control Forward
oIMR8745	-	GTC AGT CGA GTG CAC AGT TT	-	Internal Positive Control Reverse

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.51	-	4.51
10 X AB PCR BufferII	1.20	1.00 X	1.20
25 mM MgCl2	0.72	1.50 mM	0.72
2.5 mM dNTP	0.96	0.20 mM	0.96
20 uM oIMR1528	0.45	0.75 uM	0.45
20 uM oIMR1529	0.45	0.75 uM	0.45
5 mM DNA Loading Dye	1.66	0.69 mM	1.66
5 U/ul Taq DNA Polymerase	0.05	0.02 U/ul	0.05
DNA	2.00	-	2.00

Cycling

Step #	Temp °C	Time	Note
1	94	3 min	-
2	94	20 sec	-
3	64	30 sec	*-0.5 C per cycle
4	72	35 sec	repeat steps 2-4 for 12 cycles
5	94	20 sec	-
6	58	30 sec	-
7	72	35 sec	repeat steps 5-7 for 25 cycles
8	72	2 min	-
9	10	-	hold

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.81	-	4.81
10 X AB PCR BufferII	1.20	1.00 X	1.20
25 mM MgCl2	0.72	1.50 mM	0.72
2.5 mM dNTP	0.96	0.20 mM	0.96
20 ul oIMR8744	0.30	0.50 ul	0.30
20 uM oIMR8745	0.30	0.50 uM	0.30
5 mM DNA Loading Dye	1.66	0.69 mM	1.66
5 U/ul Taq DNA Polymerase	0.05	0.02 U/ul	0.05
DNA	2.00	-	2.00

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
002384	FVB/N-Tg(UcpDta)1Kz/J

Genotyping resources and troubleshooting
This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.

Cycling

Step #	Temp °C	Time	Note
1	94	3 min	-
2	94	20 sec	-
3	64	30 sec	*-0.5 C per cycle
4	72	35 sec	repeat steps 2-4 for 12 cycles
5	94	20 sec	-
6	58	30 sec	-
7	72	35 sec	repeat steps 5-7 for 25 cycles
8	72	2 min	-
9	10	-	hold