DNA Isolation from Tails - Hot Shot Method

  1. Tag and tail mice as usual.
  2. Cut a small piece of each tail (~2mm) and place in a 96-well PCR plate. Save remaining piece of tail at 4° C. Note: In step 6, it says to transfer your sample to an eppendorf, so instead of wasting money on a 96 well plate, and instead of hogging PCR machines, it may be better to just do it in eppendorf tubes that will fit in a hot block.
  3. Add 75 ul of Alkaline Lysis Buffer (see recipe below) to each sample, making sure that the tail is immersed in the buffer and that there is no air bubble at the bottom of the well. Seal plate. Important: The Tail Lysis Buffer should be prepared fresh just before adding to the tails.
  4. Put in PCR machine and remove promptly after program has finished (30 min at 95° C, followed by 15 min at 4° C. Do not leave plate in the PCR machine.
  5. Immediately add 75 ul of neutralization buffer (40 mM Tris-HCl which has not been pH’d) to the tails and mix briefly using a separate filter tip for each tail.
  6. The tail preps are now ready for PCR analysis. Use 2ul in a 20ul PCR reaction. Take up 2ul of sample from the top of the 150 ul tail prep avoiding the debris at the bottom of the well. Do not spin the tail preps in an eppendorf centrifuge because spinning can cause a proportion of the DNA to be lost in the debris at the bottom of the well thereby reducing the concentration of DNA in the sample. Wrap the plate with parafilm and store the tail preps at 4° C. For long-term storage it is best to transfer the DNA samples to 0.5ml eppendorf tubes and store them at 4° C.

Alkaline Lysis Buffer: Prepare fresh before use

Mix in 15 ml tube:

  • 10 ml sterile dWater
  • 14 ul 50% Sodium Hydroxide
  • 14 ul 0.5M EDTA, pH 8.0

Vortex Briefly

Neutralization Buffer (100ml) 40 mM Tris-HCl (not to be pH'd) pH=5 -- Filter Sterilize