CGH Nick Translation: Preparing the Reagents

CGH Nick Translation: Preparing the Reagents

 

0.2 mM SpectrumGreen or SpectrumRed dUTP

Add 10 µL of 1 mM dUTP to 40 µL nuclease-free water.

 

0.1 mM dTTP

Add 10 µL of 0.3 mM dTTP to 20 µL nuclease-free water.

 

0.1 mM dNTP mix

Mix together 10 µL each of 0.3 mM dATP, 0.3 mM dCTP, and 0.3 mM dGTP.

 

Extracted genomic DNA

Prepare a 0.2 µg/µL to 1 µg/µL solution of extracted genomic DNA in Tris-EDTA (10 mM Tris, 1 mM EDTA, pH 8.5) buffer.

 
 
 
 
CGH Nick Translation: Assay Procedure

 

This procedure labels approximately 1 µg of extracted genomic DNA. This is enough for five CGH hybridizations.

  1. Place a microcentrifuge tube on ice and allow the tube to cool.
  2. Add these components to the tube in the order listed:
    cgh-nick-translation-assay-procedure.gif
  3. Vortex the tube briefly.
  4. Incubate 2 - 4 hours at 15 °C.
  5. Stop the reaction by heating in a 70 °C water bath for 10 minutes.
  6. Chill on ice. 

Determining the Probe Size

Determining the probe size is an essential part of the CGH procedure. For detailed instruction on preparing and running an agarose gel see (1) Maniatis T, Fritsch EF, and Sambrook J. Gel electrophoresis of DNA. In: Molecularcloning: a laboratory manual. 2nd ed. Cold Spring, NY: Cold Spring Harbor Laboratory; 1989 or (2) Ausubel FM, ed. Preparation and Analysisof DNA. In: Current Protocols in Molecular Biology. New York: Greene Publishing Associates and John Wiley & Sons, 1989.

  1. Prepare a 1% agarose gel by adding 1 g agarose to 100 mL TAE buffer. Heat the solution in the microwave until the agarose is melted. This volume is enough for three minigels.
  2. Cool agarose solution to 55 °C and add 10 µL EtBr (final concentration is 0.01% (v/v)).
  3. Pour the melted agarose into a minigel (10 cm x 6.5 cm) apparatus with combs. Let the agarose cool to solidify.
  4. Pour enough TAE running buffer into the mini-gel apparatus to cover the gel by approximately 1 mm.
  5. Remove 9 µL of the reaction mix containing the nick translated DNA and add 1 µL gel loading buffer.
  6. Run the nick translated sample in one lane and a sample of the DNA size marker in another lane to size the probe.
  7. Electrophorese the gel at 10 V/cm until the dye in the gel loading buffer is 2 - 3 cm from the end of the gel.
  8. Estimate the size range of the probe from the gel. The majority of the DNA smear should be in the 300 - 3000 bp range. Probe fragments that are larger will give a diminished fluorescence intensity when used in a CGH analysis. 

As you increase the amount of enzyme and the incubation time, the size distribution shifts to progressively smaller probe fragments. To produce smaller probe fragments use these conditions that are listed in order of decreasing fragment size: 5 µL enzyme mix/2 hour incubation, 5 µL enzyme mix/4 hour incubation, 10 µL enzyme mix/2 hour incubation and 10 µL enzyme mix/4 hour incubation. Adjust the amount of nuclease-free water added to keep the total reaction volume at 50 µL.

 
 
 
 

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