Nebulization

  1. Get a nebulizer cup.  While wearing gloves, remove the plastic insert from the cup.
  2. Use scissors to trim the rigged area on the wide end of the insert.
  3. Invert the insert and place back inside the cup, sliding it down until it fits snuggly.  (Now the narrow end of the insert faces upwards.) 
  4. Set up the following in the bottom of the nebulizer cup:

50mg DNA

200ml 10X TM Buffer

1ml 80% Glycerol

Xml DIUF water

                        2ml total volume

  1. Pipette up and down to mix thoroughly.
  2. Reattach the lid to the cup.
  3. Prepare a bath of alcohol and dry ice.  Make sure it is cooling well before you use it.
  4. You will need to attach the hose of liquid nitrogen to the top spout of the cup and place the cup in the alcohol bath for the appropriate amount of time with the appropriate psi from the nitrogen for the shear size you want.

For a 2-4kb smear:   6psi for 1minute

For a 1-2kb smear:  10psi for 2 minutes

  1. Remove the cup and spin at 5000rpm for 1min to pull as much as possible to the bottom of the cup.
  2. Transfer 400ml to as many 1.5ml sterile microfuge tubes as needed (usually 5 tubes, and q.s. the 5th tube up to 400ml with DIUF water if you couldn’t retrieve the full amount).
  3. Add 40ml 3MnaOAc pH 5.1 to each tube.
  4. Flick the tube well to mix.
  5. Add 880ml 95% alcohol and flick well to mix.
  6. Put at –80°C overnight and finish the ethanol precipitation the following day.  You could wait longer if you’re having trouble recovering DNA.

To finish ethanol precipitation:

  1. Spin at 13,000 rpm, 4°C, for 1hour.
  2. Dump the supernatant (and save in case you lose DNA).
  3. Wash with 400ml cold 70% alcohol.
  4. Spin at 13,000rpm, 4°C, for 30 minutes.
  5. Dump and save.
  6. Spin for another 2 minutes to bring down any excess alcohol, then remove with pipetter.
  7. Dry the samples at room temp. or in the spin vac.
  8. Resuspend with a total volume of 50ml sterile water.  (Resuspend one tube, transfer to next and resuspend, etc.)  If you have trouble recovering DNA, warm tubes to 50°C or use more water in resuspension.
Now the DNA is ready to be checked on a gel and to have the OD read.