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Method: Purification and Sequencing of PCR Product

May 11, 1990

Terry C. Lairmore


Principle:

Time required:

Special reagents:

Special equipment:

Procedure:

  1. Reduce total volume of the PCR reaction product (50 Ál) to approximately 25 Ál by placing in a vacuum dessicator for 30-60 minutes.
  2. Pre-spin the Linkers 6 quick-spin Sepharose columns CL-6B (place columns within a 15 ml snap cap to spin), for 5-10 minutes at 1,100 x g in a swinging bucket rotor to get rid of excess buffer and discard the flow-through. Change the collection tube and pre-spin a second time for 5-10 minutes at 1,100 x g and again discard the buffer.
  3. Load 25 Ál (maximum volume for the columns) directly into the center of each column and spin at 1,100 x g for 10 minutes in Beckman TJ-6 (approximately 2500 rpm), collecting the product in a clean labeled tube. Yield is approximately 35-50 Ál.
  4. Remove 1/5 of purified product to a clean tube. To denature the DNA in the aliquot add 0.2 N NaOH (to total volume of 40 Ál), mix and hold at room temperature for 5 minutes.
  5. Neutralize the solution with 1/3 volume (13.3 Ál) of 3 M KOAc and 2.5 volumes (100 Ál) of 100% ethanol.
  6. Chill to -20 degrees C for 20 minutes, then pellet the precipitate with a 15 minute, 12,000 rpm spin in a 4 degrees C microcentrifuge.
  7. Wash the pellet with 70% ethanol and air dry or dry in a vacuum dessicator.

Sequencing may be carried out using the SequenaseR kit and a slight modification of the double stranded sequencing protocol:

  1. Resuspend pellet (not always visible) in:

  2. Place tube at 37 degrees C for 15 minutes for annealing of primer and template.
  3. Add 2 Ál diluted labeling mix (1:5 to 1:15)

  4. Dispense 3.5 Ál into each of 4 prelabeled tubes (G, A, T, C) containing 2.5 Ál of appropriate termination mix (ddGTP, ddATP, ddTTP, ddCTP) and incubate tubes at 37 degrees C for 5 minutes or longer (volume of each tube is 6 Ál).
  5. Add 4 Ál of Sequenase stop solution to each tube. Samples may be stored at -20 degrees C until they are run on a gel.
  6. Heat the samples to 75-80 degrees C for 2 minutes and load 1-3 Ál per lane immediately onto a sequencing gel.
Reference:

method after B. Dubose (pers. comm., Hartl lab)