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Protocols for DNA Marker Labeling:

• Radioactive DNA Ladder/Marker Labeling using T4 Polynucleotide Kinase (see below)

• Radioactive DNA Ladder/Marker Labeling by Filling-in 3'-recessed Termini using Klenow Fragment, exo-

Radioactive DNA Ladder/Marker Labeling using T4 Polynucleotide Kinase
The ready-to-use versions of the DNA ladders/markers can not be radiolabeled with T4 Polynucleotide Kinase

1. Prepare the following reaction mixture:

   DNA ladder/marker	1-20 pmol* of 5'-termini
   10X buffer B for exchange reaction (supplied with T4 Polynucleotide Kinase)	2 µl
   [gama-32P]-ATP or [gama-33P]-ATP	40 pmol
   24% (w/v) PEG 6000 solution (supplied with T4 Polynucleotide Kinase)	4 µl
   Water, nuclease-free	to 19 µl
   T4 Polynucleotide Kinase	1 µl (10 u)
   Total volume:	20 µl

2. Mix well. Incubate at 37°C for 30 minutes.
3. Add 1 µl of 0.5 M EDTA (pH 8.0) and extract with an equal volume of chloroform.
4. Purify labeled DNA on a Sephadex G-50 column. Determine the efficiency of the label incorporation if necessary.

Note

• All types of DNA ends can be successfully labeled with T4 Polynucleotide Kinase. However, the labeling efficiency is greatest for the 5'-protruding DNA ends. The efficiency is lower for blunt ends, and it is lowest for 5'-recessed DNA ends.
• If an ethanol solution of [gama-³²P]- or [gama-³³P]-ATP is used, first dry the required quantity of ATP under vacuum, then dissolve the residue in deionized water.
• Final ATP concentration should be at least 2 µM. If [gama-³²P]-ATP with a high specific activity (higher than 5000 Ci/mmol) is used, the label can be diluted with ATP.

* estimation of DNA markers 5'-ends concentration: pmol ends = pmol DNA x number of bands x 2

Ordering Information

T4 Polynucleotide Kinase GeneRuler™ DNA Ladders Conventional Lambda DNA Markers Conventional Phage and Plasmid DNA Markers ATP 0.5 M EDTA (pH 8.0) Water, nuclease-free

Radioactive DNA Ladder/Marker Labeling by Filling-in 3'-recessed Termini using Klenow Fragment, exo-
The ready-to-use versions of the DNA ladders/markers can not be radiolabeled with Klenow Fragment, exo-

1. Prepare the following reaction mixture:

   DNA ladder/marker	1-2 µl (0.5-1 µg)
   10X reaction buffer (supplied with Klenow Fragment, exo-)	2 µl
   [alfa-32P]-dNTP, ~15-30 TBq/mmol (400-800 Ci/mmol) or [alfa-32P]-dNTP, ~110 TBq/mmol (3000 Ci/mmol)	0.74 MBq (20 µCi) 2.96 MBq (80 µCi)
   3 dNTP Mix, 2 mM each (without a labeled dNTP)	2.5 µl (0.25 mM final concentration)
   Klenow Fragment, exo-	1 u
   Water, nuclease-free	to 20 µl

2. Incubate at 30°C for 15 minutes.
3. Stop the reaction by heating at 70°C for 10 minutes.

Note

The choice of ³²P-labeled dNTP depends on the nucleotide sequence of the protruding 5' termini which serves as a template for filling-in 3'-recessed DNA ends.

Ordering Information

Klenow Fragment, exo- Conventional Lambda DNA Markers Conventional Phage and Plasmid DNA Markers Water, nuclease-free

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Updated gegužės 10, 2007 13:06