Jacobs:Protocol 10 bp DNA Ladder

Overview

• Concentration: 1 μg/μL
• Size: 50 μg
• Store at -20°C

The 10 bp DNA Ladder consists of thirty-three 10-bp repeats plus a fragment at 1668 bp and is suitable for sizing both double-stranded and single-stranded DNA fragments from 10 bp to 200 bp. The 100-bp band is approximately two to three times brighter than other ladder bands to provide internal orientation. In addition, because both DNA strands are of the same nucleotide composition, this product can be denatured to produce a set of single-stranded oligonucleotides increasing in length by 10-nucleotide increments. The double-stranded ladder can be visualized on 4% to 5% agarose gels after ethidium bromide staining. The single-stranded ladder can be visualized on an 8% urea-polyacrylamide gel after electrophoresis. This ladder can be easily radiolabeled using T4 polynucleotide kinase.

Materials

• Storage Buffer:
  • 10 mM Tris-HCl (pH 7.5)
  • 1 mM EDTA

Procedure

For agarose gel electrophoresis, apply approximately 0.4 μg of ladder per mm lane width. Do not heat before loading. Incorporate ethidium bromide (0.5 μg/ml) into both the gel and running buffer to examine the gel directly under UV-illumination and avoid diffusion of the bands.

• Note: During Low Melting Point agarose gel electrophoresis (4% LMP agarose) with Tris-acetate

(pH 7.6) as running buffer, bromophenol blue migrates between the 20-bp and 30-bp bands. The 100-bp band will appear two to three times brighter than other bands.

Labeling of the 10 bp DNA Ladder with 32P:

5′P---3′OH + [γ-32P]ATP + ADP T4 polynucleotide>5′P32---3′OH + ATP + ADP kinase

1. Dilute the 10 bp DNA Ladder stock ten-fold with TE to a final concentration of 0.1 μg/μl.
2. Pipet into a 0.5-ml microcentrifuge tube on ice:
   1. 2 μl diluted ladder
   2. 1 μl 5X Exchange Reaction Buffer [250 mM imidazole(pH 6.4), 60 mM MgCl2, 5 mM 2-mercaptoethanol, 350 μM ADP]
   3. 1 μl γ32P ATP (>5,000 Ci/mmole, 10 μCi/μl)
   4. 1 μl T4 polynucleotide kinase (10 U/μl)
3. Make sure all components are at the bottom of tube. Mix thoroughly but not vigorously. Centrifuge briefly.
4. Incubate for 10 min at 37°C.
5. Stop the reaction by heating the tube 5 min at 55°C.
6. This reaction will yield specific activities of approximatelyb 2 × 106 cpm/μl.

Generating a single-stranded 10 nucleotide ladder:

1. To 5 μl of 10 bp DNA Ladder, add an equal volume of denaturing solution [95% (v/v) formamide, 10 mM EDTA (pH 8.0), 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol].
2. Incubate at 70°C for 5 min.
3. Electrophorese the sample in a denaturing 8% polyacrylamide/urea gel (Gel-Mix 8 is recommended). A set of single-stranded oligonucleotides (10-200 nucleotides) increasing in length by 10-nucleotide increments should be clearly visible after ethidium bromide staining.

Quality Control:

Agarose gel analysis shows that the bands between 10 bp to 200 bp are distinguishable. The 100-bp band must be more intense than bands ranging from 10 bp to 330 bp. Upon denaturing of the 10 bp DNA Ladder with formamide and subsequent gel electrophoresis, a set of single-stranded oligonucleotides is clearly visible after ethidium bromide staining.

Contact

• Cat. No. 10821-015, revised on 10/10/01

or instead, discuss this protocol.