Knock Out Candidates: Frozen Stocks, Genomic DNA, and PCR

In this protocol you take a confluent plate of cells, make three aliquots of frozen stocks, and use the remainder of the cells to prepare genomic DNA for PCR

Prepare Frozen Stocks

  1. REsuspend a 100mm (10mL) plate of cells that is a bit past confluent (a few floaters)
  2. Transfer cells to a 15ml Falcon Tube and spin @ 100rpm for 5min in centrifuge (Steen's).
  3. Aspirate off supe, and resuspend cells in 2.5ml of MES-buffered HL-5.
  4. Add 2.5ml MES-buffered HL-% plus 20% DMSO, and pipet up and down 3-4x to mix.
  5. Transfer 250ul to three labeled tubes. (Use automatic pipetor)
  6. Place tubes in RT "Mr Frosty" (Filled w/ methanol to line. Can use methanol 3x)
  7. Place "Mr. Frosty" in -80C freezer. Cooling occurs at 1C per minute, so after 3hrs the tubes should reach -80C. They can be transfered to a storage box anytime after 2hrs.
  8. Make DNA from remaining Cell Suspension

Prepare Genomic DNA

  1. Spin down cells as above (1000rpm/5min)
  2. Aspirate of sup and resuspend cells in 1ml DNAzol using cut-tip p1000.
  3. Transfer DNAzol/cell solution to an Epe. tube. Spin at 11k rpm (5-10min) in minifuge at Rt or 4C.
  4. Transfer the supe to a screw-cap tube. add 0.5ml 100% Ethanol and mix the samples by inverting several times. Incubate @RT for 1-3min.
  5. Spin for 2min @ 7k rpm at RT in minifuge. Pour off supe, and wash pellet twice w/ 1ml 75% ethanol/ After last wash, spin tubes again and remove remaining ethanol w/ a p200.
  6. Add 200ul TE to each tube, and gently pipet up and down to disrupt pellet. Then add 0.4ul 10mg/ml RNAse. Or use 200ul of TE/RNase mini-prep mix.
  7. Store at 4C
  8. Spin 10min at max speed in minifuge and use 5ul of supe in PCR.
  9. PCR and run on agarose gel.