Product Literature Vol 3 Optimax Genotyping | Page 2

Transgenic Mouse

PCR set up

Electrophoresis

The three transgenes of

A master mix was prepared

Following the PCR reaction,

interest in the PTD mouse

based on the amount of

12.5 μL of the samples

line are the P (promoter), T

PCR reactions performed

were loaded onto a 2%

(F1/F1 – floxed allele), and

during each study. To

agarose gel (BioExpress,

D (reporter allele) modified

prepare the mix, 62.5 μL of

Cat No. 0710-100G)

endogenous genes. The

OneTaq® 2X Master Mix

containing 10 μL of

selected PCR primers for

with Standard Buffer (New

Ethidium bromide (Fisher,

each have annealing

England Biolabs, Cat. No.

Cat. No. BP902-1) in 1X

temperatures of 51.7°C,

M0482L), 5 μL of each

TAE (Fisher, Cat. No.

65.0°C, and 61.0°C,

primer, and 27.5 μL of H2O

BP902-1). Additionally, 15

respectively.

was added to a

μL of the Quick-Load® Low

DNA extraction

microcentrifuge tube. For

Molecular Weight DNA

For the DNA extraction, 1

each PCR reaction, 25 μL

Ladder (New England

mm of mouse tail was

of the master mix was

Biolabs, Cat. No. N0474S)

D Gene

excised, added to 75 μL of

aliquoted into a 0.2mL PCR

was also loaded onto the

94°C for 3 minutes
35 cycles of:
94°C for 30 seconds
61.0°C for 1
68°C for 1 minute
Then
68°C for 2 minutes
4°C hold

alkaline lysis buffer (25 mM

tube (BioExpress C-3310-1)

gel. The gel was transferred

NaOH, 0.2 mM EDTA, pH

When programming the

to an electrophoresis unit ,

12) and vortexed for 1

MultiGene OptiMax, Blocks

which was operated under

minute. The extract mixture

1 2, and 3 were assigned

constant voltage at 100

was then incubated for 30

for the P, D, and T genes

volts for 1 hour. Following

minutes at 37°C and

respectively. The ramp rate

electrophoresis, the gel was

vortexed briefly. Lastly, 75

on the MultiGene OptiMax

imaged using an Enduro

μL of the neutralizing

is 5°C /sec heating and

GDS (Labnet, Cat No.

solution (40 mM Tris-HCl,

3.5°C /sec cooling.

GDS-1302).

P Gene
94°C for 3 minutes
35 cycles of:
94°C for 30 seconds
51.7°C for 1 minute
68°C for 1 minute
Then
68°C for 2 minutes
4°C hold

T Gene
94°C for 3 minutes
35 cycles of:
94°C for 30 seconds
65.0°C for 1 minute
68°C for 1 minute
Then
68°C for 2 minutes
4°C hold

pH 5) was added to the
DNA extraction tube and
briefly vortexed again.