Master Protocol Details

Notes:

Melting curve analysis is done using a Roche Light Cycler 480.

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

Expected Results:

Transgene Tm = 87°C +/- 1°C
Internal positive control Tm = 79-80°C+/- 1°C

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
oIMR7338	-	CTA GGC CAC AGA ATT GAA AGA TCT	-	Internal Positive Control Forward
oIMR7339	-	GTA GGT GGA AAT TCT AGC ATC ATC C	-	Internal Positive Control Reverse
oIMR9266	-	AGA TGC CAG GAC ATC AGG AAC CTG	-	Transgene Forward
oIMR9267	-	ATC AGC CAC ACC AGA CAC AGA GAT C	-	Transgene Reverse

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	1.64	-	1.64
25 mM MgCl2	0.96	2.00 mM	0.96
20 uM oIMR9266	0.10	0.17 uM	0.10
20 uM oIMR9267	0.10	0.17 uM	0.10
20 uM oIMR7338	0.60	1.00 uM	0.60
20 uM oIMR7339	0.60	1.00 uM	0.60
DNA	2.00	-	2.00
10 X High Resolution Melt Master Mix (Roche)	6.00	5.00 X	6.00

Cycling

Step #	Temp °C	Time	Note
1	94	10min	-
2	94	30 sec	-
3	62	1 min	-
4	72	30 sec	repeat steps 2-4 for 35 cycles
5	72	2 min	-
6	10	-	hold

This genotyping protocol is used for the following strains:

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