Master Protocol Details

Expected Results:

Melt curves
Wt = lower, Het = middle, Hom = upper

Mutant = 312 bp
Heterozygote = 137 bp and 312 bp
Wild type = 137 bp

Gel Image

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Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
oIMR7376	-	CCA CAC GCG TCA CCT TAA TA	-	Mutant
oIMR9573	-	CTT GCA CTA CCA AAG CCA CA	-	Common
oIMR9574	-	GTT ATT GTC TTC CCG GCT GT	-	Wild type

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	3.65	-	3.65
5 X Kapa 2G HS buffer	2.40	1.00 X	2.40
25 mM MgCl2	0.96	2.00 mM	0.96
10 mM dNTP KAPA	0.24	0.20 mM	0.24
20 uM oIMR7376	0.30	0.50 uM	0.30
20 uM oIMR9573	0.30	0.50 uM	0.30
20 uM oIMR9574	0.30	0.50 uM	0.30
5 mM 10x Loading Dye	1.20	0.50 mM	1.20
2.5 ul Kapa 2G HS taq polymerase	0.05	0.01 ul	0.05
20 X EvaGreen	0.60	1.00 X	0.60
DNA	2.00	-	2.00

Cycling

Step #	Temp °C	Time	Note
1	94	2 min	-
2	94	20sec	-
3	65	15sec	-1.5 C per cycle
4	68	10sec	-
5	-	-	repeat steps 2-4 for 10 cycles
6	94	15sec	-
7	50	15sec	-
8	72	10sec	-
9	-	-	repeat steps 6-8 for 28 cycles
10	72	2 min	-
11	10	-	hold

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
002250	B10.129P2(B6)-Il10^tm1Cgn/J
002251	B6.129P2-Il10^tm1Cgn/J
003968	C3Bir.129P2(B6)-Il10^tm1Cgn/LtJ
004228	NOD.Cg-Il10^tm1Cgn Il4^tm1Cgn/Dvs
004266	NOD.Cg-Il10^tm1Cgn/DvsJ
004291	NOD.Cg-Il10^tm1Cgn Il4^tm1Cgn/DvsJ
004326	C3Bir.129P2(B6)-Il10^tm1Cgn/Lt
004333	C.129P2(B6)-Il10^tm1Cgn/J
004368	129(B6)-Il10^tm1Cgn/J
005346	NOD.Cg-Il10^tm1Cgn Casp1^tm1Sesh Casp4^del/LtJ
005912	B6.Cg-Il10^tm1Cgn (D3Mit49-D3Mit348)/Lt

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