Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The material on this page is part of Chapter 10, which is shown in full as a preview on this site.

Chapter 10: Nucleic Acid Platform Technologies

Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

INTRODUCTION

Indirect Labeling of DNA

(Protocol summary only for purposes of this preview site)

As with RNA labeling protocols, the main difference between direct and indirect DNA labeling protocols is a trade-off of cost and time. Indirect labeling of DNA takes 2 h longer than direct labeling but is hundreds of dollars cheaper.


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 Protocol 8: Indirect Labeling of DNA doi10.1101/molclon.000186

As with RNA labeling protocols, the main difference between direct and indirect DNA labeling protocols is a trade-off of cost and time. Indirect labeling of DNA takes 2 h longer than direct labeling but is hundreds of dollars cheaper.

MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.

Reagents

  • aa-dUTP/dNTP mixture (3 mM)
    • Combine 6 L of 50 stock <R> used for cDNA synthesis and 44 L of dH2O.
  • Cyanine dyes (typically Cy5 and Cy3) (GE Healthcare Life Sciences)
    • Dyes come dried and should be resuspended in 11 L of DMSO. This amount of material is sufficient for three labeling reactions. If the entire amount will not be used, prepare 3-L aliquots, dry down in a rotary vacuum, and store them at 20C.
  • EDTA (0.5 M, pH 8.0)
  • Genomic DNA
    • Prepare genomic DNA according to your favorite protocol. Because the DNA should be fairly pure, avoid using quick and dirty methods. Commercial gDNA isolation kits are suitable, as in Chapter 1, Protocols 12 and 13. As a rule of thumb, the OD260/280 ratio should be at least 1.8.
  • Klenow buffer
  • Klenow fragment of DNA polymerase I
  • NaHCO3 (50 mM, pH 9.0)
  • Random hexamer
    • N6 random hexamer can be ordered from any oligonucleotide company, made up at 5 g/L in RNase-free TE/H2O.

Equipment

  • Heat block set at 37C and 100C
  • Lightproof box (see Step 11)
  • MinElute kit (QIAGEN)
  • Zymo columns (Zymo Research, catalog no. D3024)

METHOD
Genomic DNA Labeling

  • 1. For each array, combine 4 g of genomic DNA, 10 g of random hexamer (N6), and dH2O to a final volume of 42 L. Incubate for 5 min at 100C.
  • 2. Cool the tube(s) quickly on ice for 10 min.
  • 3. Pulse-centrifuge to bring the condensate to the bottom of the tube.
  • 4. On ice, add:
    Pipette to mix. Incubate for 2 h at 37C.
  • 5. Add 5 L of 0.5 M EDTA (pH 8.0) to stop the reaction.

Purification of Random Prime-Labeled DNA

  • 6. Add 1 mL of Binding buffer to the reaction. Mix well, and load half (527 L) of the reaction mixture onto each of two Zymo columns. Centrifuge the columns for 10 sec at maximum speed.
    • The large volume of Binding buffer is necessary to precipitate single-stranded DNA.
  • 7. Discard the flowthrough. Wash the column with 200 L of Wash buffer. Centrifuge for 1 min at maximum speed.
  • 8. Repeat Step 7.
  • 9. Add 10 L of 50 mM NaHCO3 (pH 9.0) to each column filter. Incubate for 5 min at room temperature.
  • 10. Elute the DNA from the column by centrifuging for 1 min at maximum speed.
  • 11. Couple cyanine dyes to the aa-dUTP-incorporated cDNAs by adding the eluted material directly to 3 L of Cy5 dye or Cy3 dye resuspended in DMSO. Incubate for 1 h to overnight at room temperature in a lightproof box or a drawer.

Purification of Labeled Genomic DNA

  • 12. Purify the cDNA using a MinElute kit, following the manufacturer's instructions.

RECIPE

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

aa-dUTP/dNTP Mixture (50)

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