Method: Labeling DNA with 32P-dCTP by Primer Extension

June 22, 1990

Srini Ramachandra

Principle:

Double stranded DNA is heat-denatured and random hexanucleotide primers are attached to the single strand. The synthesis is carried out using Klenow fragment of DNA polymerase I incorporating 32P-dCTP at the same time. The Klenow enzyme lacks the 5'----> 3' exonuclease activity, so that the radioactive product is synthesised exclusively by primer extension rather than nick translation. The reaction is carried out at pH6.6 thereby reducing the 3' ----> 5' exonuclease activity of the Klenow enzyme. On an average 35-40% incorporation of 32P-dCTP can be expected. A 50 ng reaction will usually generate 5 x 107 dpm. Ideally we need 1 - 5 x 107dpm of labeled probe DNA per blot (20cm x 20cm) and 0.5 - 2.5 x 107dpm of labeled lambda DNA per blot. If more than 5 blots are used per probe all components of the reaction can be doubled or tripled, but the volume of TE applied to the column must be reduced to maintain the volume of labeled probe at or below 500 µl.

Time required:

2 hours

Procedure:

Note: All volumes are for a 50 ng reaction. The reaction volume can be scaled up to label 150 ng of DNA per tube, scaling up all volumes to 3 times the original volume.

Labeling the probe:

1. Thaw out 32P-dCTP on a fresh piece of absorbent pad behind the radioactive shield for at least 30 minutes. (But do not let it sit at room temperature for hours, as the nucleotide will start to breakdown).
2. To an eppendorf tube add the following:

   12.5 ng/µl DNA stock	= 4 µl (50ng)
   sterile ddH2O	= 3 µl
   	-------
   	7 µl

3. Place the tube in the metal holder and heat the DNA for 2 minutes in a boiling water bath, maintaining a temperature of 95 degrees C.
4. Quick-cool the tubes on ice for 2 minutes and touch-spin in a microcentrifuge to bring down the condensation on the sides.
5. Add the following in the order given:

   LS-C	= 11.4 µl	(If using 32P-dATP, you will have to use LS-A.)
   10mg/ml BSA	= 1.0 µl
   Klenow enzyme	= 1.0 µl
   	--------
   	20.4 µl

6. Add 5 µl of 32P-dCTP to the tube and mix the contents well. Work behind a plexiglass shield in the radioactive work area.
7. Incubate the reaction mix on a 37 degrees C heat block for 30 minutes to an hour.

P-60 Column preparation:

Prepare the Bio-gel P60 columns as follows: cut the Kontes straw columns into halves. Place the disc filter in place in the conical adaptor and attach the the adaptor to one end of the straw column. Place the empty columns on a single-row plexiglass rack and a large weigh boat as a drain pan below. Add enough P60 slurry (made in TE) to the columns and gently tap the columns against the rack to allow the TE to drip through. You can also start the flow of TE by putting a gloved index finger over the column opening (to seal it from the atmosphere), then gently squeeze the column below. When the column starts dripping, remove the index finger first, then release the pressure from the sides of column (avoid drawing air up through the bottom of column). Pack the column to about 1 cm above the column rack level. Fill column with fresh TE and allow to drip dry. Cut off the excess column about 1 - 1.5 cm above the P60 surface.

Probe purification:

1. Stop the reaction by adding 17µl of 10X Labeling Stop Mix (contains Dextran blue and Bromophenol blue dyes) to each tube. Add the reaction mix drop by drop to the top of the column without disturbing the surface and let the excess TE drip through the column into a weigh boat or a steel pan.
2. Add enough TE to the top of the column to move the dextran blue (the light blue dye) to within 1cm of the conical tip of the column (~300 µl TE; allow the TE to drip into a weigh boat). Start collecting the probe approximately 1cm before the dextran blue starts coming off the column: first placing the microcentrifuge tubes (in a Sarstedt rack) under the columns, then add TE to each column. To accomodate the Probe Count, the collected volume of labeled probe, and therefore the added volume of TE, should be either 450 µl or 500 µl. Do not collect any of the dark blue dye (Bromophenol blue). It contains the unincorporated nucleotides.
3. Place the used columns into a used 50ml centrifuge tube and discard all dry waste into the radioactive dry waste container. Soak the radioactive drips in weigh boat with Kimwipes and discard in radioactive dry waste container.
4. Follow the instructions on page 9 of the ONCOR Probe Count instruction manual and determine the dpms (disintegrations per minute) of the labeled probes.
5. Use the labeled probes immediately for hybridization as described under "Hybridization to Southern Blots".

Solutions:

• AGT combo
  dATP, dGTP & dTTP in the Ultrapure dNTP set (Pharmacia, Cat.#27-2035-01) are supplied in 100 mM concentrations. Make up 300 µM dilutions of each nucleotide in TM as follows: 3 µl of 100 mM stock in 1ml of TM will give a 300 µM dilution of each.

  For AGT combo: mix 1:1:1 volumes of each of the 300 µM stocks to get a final concentration of 100 µM each of dATP, dGTP and dTTP. Store all stock solutions at -20 degrees C.

• Oligo Labeling solution (OL)

  The final concentration of OL is 0.1 U/µl. pd(N)6 (Pharmacia, Cat.# 27-2166-01) is supplied at 50 units/bottle. Add 500 µl of 1mM Tris.HCl, 1mM EDTA (pH7.5) directly to the bottle and dissolve the contents thoroughly (goes into solution very easily). Transfer to a sterile eppendorf tube labeled OL and store at -20 degrees C.

• 1mM Tris.HCl, 1mM EDTA

  Add 100 µl 1M Tris.HCl, pH 7.5 and 200 µl 0.5M EDTA, pH 8.0 to 100 ml of ddH2O,

  filter sterilize and store at 4 degrees C.

• BSA

  Make 10 ml of 10 mg/ml dilution from a 50 mg/ml BSA stock with sterile ddH2O,

  aliquot 1 ml into eppendorf tubes and store at -20 degrees C.

• LS-C

  Mix:	(25 parts) 1M HEPES, pH 6.6:	1.0 ml
  	(25 parts ) AGT combo:	1.0 ml
  	(7 parts) OL solution:	280 µl
  	Store at -20 degrees C in 100µl aliquots in eppendorf tubes.

• TM:

  		Final concentration
  1M Tris.HCl, pH8	2.5 ml	250 mM
  1M MgCl2	250 µl	25 mM
  Beta-Mercaptoethanol (12M)	42 µl	50 mM

  Adjust volume to 8.0 ml with ddH2O

  pH to 8.0, bring volume to 10ml, filter sterilize and store at 4 degrees C.

• 1M HEPES, pH 6.6
  dissolve 28.33 g of HEPES (Sigma) in ddH2O

  Adjust volume to 100 ml, filter sterilize and store at 4 degrees C.

• Bio-Gel P60

  Use Coarse, mesh size 50-100, preswollen (Bio-Rad Cat.# 150-1630)

  Transfer the entire contents of the Bio-Gel P60 bottle (100g) into a 4 liter flask or a Nalgene beaker, add 1500ml or more of TE, mix well and let it sit at room temperature overnight. Decant the fines with the TE and add fresh TE, mix and store at room temperature.

• 10X Labeling Stop Mix

  32.0 ml sterile dd H2O

  2.0 ml 0.5M EDTA, pH 8.0

  1.0 ml 10 mg/ml E. coli tRNA

  0.70 g Blue Dextran

  0.07 g Bromophenol Blue

  Store at 4 degrees C.

• E. coli tRNA (Sigma, Cat.#R-7501)

  Prepare 10 mg/ml in sterile ddH2O, and store at -20 degrees C.

References:

Feinberg, A.P. and B. Vogelstein. (1983). "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity" Anal. Biochem. 132: p. 6-13.

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