Random Primer DNA Labeling

Objective:

To produce radioactively labeled DNA strands for the detection of target DNA or RNA sequences in various applications including Southern and northern blot hybridization.

Required Materials

  • GibcoBRL Random Primer Labeling Kit
    • Random primers buffer mixture
    • dATP solution
    • dCTP solution
    • dGTP solution
    • dTTP solution
    • Klenow Fragment (Large Fragment of DNA Polymerase I)
    • Stop Buffer
    • Control DNA
  • dsDNA Fragment
  • Distilled Water
  • [a-32P]dCTP
  • Radioactivity monitoring and waste management equipment
  •  1 mL syringe
  •  Sephadex G-50 saturated in TE buffer pH 7.4, 0.2% SDS.

Procedures

Start=> You must first have a purified dsDNA fragment of known concentration that you wish to label.

  1. Combine the following:
    • 25 ng dsDNA fragment in 5-20uL dH2O
    • 2 uL dATP solution
    • 2 uL dGTP solution
    • 2 uL dTTP solution
    • 15 uL Random Primer Buffer Mixture
    • 5 uL [a-32P]dCTP (approximately 50 uCi)
    • Distilled water to a volume of 49 uL
  2. Mix briefly, and pulse down in a microcentrifuge.
  3. Denature dsDNA by boiling for 5 minutes. Then immediately place on ice.
  4. Add 1 uL Klenow Fragment, mix gently but thoroughly. If necessary, briefly pulse down contents in a microcentrifuge.
  5. Incubate at room temperature (25 degrees Celsius) for 1 hour.
  6. Add 2 uL of stop buffer.

 

To purify and/or determine the activity of incorporated dCTP- continue.

  1. Dilute 2 uL of the 52 uL sample into 500 uL with 498 uL of distilled water for use later when determining total radioactivity.
  2. Place a small amount of glass wool at the bottom of a 1 mL syringe.
  3. Load sephadex G-50 (saturated inTE pH 7.4, 0.2% SDS) into the 1 mL syringe.
  4. Pack the mini-column by spinning the 1 mL syringe in a 15 mL conical tube in a clinical centrifuge on a setting of 6 for 30 seconds.
  5. Repeat steps 9 and 10 until the column fills the syringe to approximately 0.9 mL.
  6. Add the remaining 50 uL of the labeling reaction onto the column. Rinse the labeling reaction with 50 uL TE pH 7.4 and add the rinse to the column.
  7. Place a clean 1.5 mL microcentrifuge tube at the bottom of the 15 mL conical tube to collect your sample.
  8. Spin the column in the clinical centrifuge for one minute on setting 6.
  9. Add an additional 100 uL of TE pH 7.4 to the column and spin down for one minute at setting 6.
  10. Check the volume of the collected sample and adjust to 200 uL if short.
  11. Dilute 2 uL of the 200 uL sample into 125 uL by adding 123 uL of distilled water.
  12. Label two Whatman GF/C filters, total and inc. Then spot the total filter with 5 uL of diluted probe from step 7 and spot the inc. filter with 5 uL of the diluted and purified probe from step 17.
  13. Allow the filters to dry or dry under a heat lamp.
  14. Check radioactivity in a liquid scintillation counter.
    • Total radioactivity(cpm) is equal to 2600 x cpm[total]
    • Incorporated radioactivity(cpm) is equal to 2500 x cpm[inc]
    • % incorporated is equal to (inc/50)/(total/52)*100
  15. Store the probe at -20 degrees.