Master Protocol Details



Notes:	This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Expected Results:	Transgene = 264 bp Internal positive control = 200 bp

Gel Image

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

Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
oIMR6501	-	CCA TCC ACG CTG TTT TGA CCT C	-	Transgene
oIMR6502	-	TAC TCC TCT TGA CCT GCT GTG TCG	-	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	4.56	-	4.56
10 X PCR Buffer II (ABI)	1.20	1.00 X	1.20
25 mM MgCl2	0.96	2.00 mM	0.96
2.5 mM dNTP	0.96	0.20 mM	0.96
20 uM oIMR6501	0.30	0.50 uM	0.30
20 uM oIMR6502	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	0.06	0.03 U/ul	0.06
DNA	2.00	-	2.00

Cycling

Step #	Temp °C	Time	Note
1	94	3 min	-
2	94	30 sec	-
3	50	1 min	-
4	72	1 min	repeat steps 2-4 for 35 cycles
5	72	2 min	-
6	10	-	hold

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	4.56	-	4.56
10 X PCR Buffer II (ABI)	1.20	1.00 X	1.20
25 mM MgCl2	0.96	2.00 mM	0.96
2.5 mM dNTP	0.96	0.20 mM	0.96
20 oIMR8744	0.30	0.50	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	0.06	0.03 U/ul	0.06
DNA	2.00	-	2.00

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
006439	FVB-Tg(tetO/CMV-KRAS*G12C)9.1Msmi/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	30 sec	-
3	62	1 min	-
4	72	1 min	repeat steps 2-4 for 35 cycles
5	72	2 min	-
6	10	-	hold