Biotin Labeling

Manufacturer’s instructions

Materials needed

Reagent	Producer	Catalog #
DecaLabel™ Biotin DNA Labeling Kit	Thermo Scientific	FERK0651

Procedure

1. Add the following components into 1.5 ml microcentrifuge tube:

Component	Volume
RE and MBN treated DSN Library	20 ul
Decanucleotide in 5X Reaction Buffer	10 ul
Water, nuclease-free	14 ul
Total Volume	44 ul

RE stands for Restriction Enzyme and MBN stands for Mung Bean

2. Vortex the tube and spin down in a microcentrifuge for 3-5 seconds.
3. Incubate the tube in a boiling water bath for 5-10 min and cool it on ice. Spin down quickly.
   

1. Add the following components in the same tube:

Component	Volume
Biotin Labeling Mix	5 ul
Klenow fragment, exo– (5 u)	1 ul
Total Volume	50 ul

2. Shake the tube and spin down in a microcentrifuge for 3-5 seconds.
   
3. Incubate for 12-20 hours at 37°C on heat block.

3. Stop the reaction by the addition of 1 μl of 0.5 M EDTA, pH 8.0.

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Optional:Control reaction

1. Add the following components into 1.5 ml microcentrifuge tube:

Component	Volume
Control Template, 10 ng/ul	25 ul
Decanucleotide in 5X Reaction Buffer	10 ul
Water, nuclease-free	9 ul
Total Volume	44 ul

• Vortex the tube and spin down in a microcentrifuge for 3-5 seconds
• Incubate the tube in a boiling water bath for 5-10 min and cool it on ice. Spin down quickly in microcentrifuge.

2. Add the following components in the same tube:

Component	Volume
Biotin Labeling Mix	5 ul
Klenow fragment, exo– (5 u)	1 ul
Total Volume	50 ul

2. Shake the tube and spin down in a microcentrifuge for 3-5 seconds.
   
3. Incubate for 12-20 hours at 37°C on heat block.

3. Stop the reaction by the addition of 1 μl of 0.5 M EDTA, pH 8.0.