Posted on May 16th, 2016 by UNM CC
Cell lysis buffer: 10 mM Tris pH8, 100 mM NaCl, 25 mM EDTA, 0.5% (w/v) SDS, 0.5 mg/mL proteinase K
Fixative: 1 volume glacial acetic acid + 3 volumes methanol
75% EtOH made with RNAse free H2O
Phenol: Buffered to pH=8 with Tris.
Chloroform: Stabilized with isoamyl alcohol.
T.E.: 10 mM Tris pH8, 1 mM EDTA.
This protocol prepares genomic DNA from cryopreserved leukemia cells. It assumes a starting volume of ~1.8 mL and uses only 1/2 of this volume. The remainder is frozen again. The DNA preparation protocol does not contain a RNAse step. It may be necessary to add this if a significant amount of RNA contaminates the DNA. It also saves an aliquot of RNA from each sample, partially purified by the Trizol protocol. A third aliquot is used to fix cells for cytology.
- Place freezer boxes on dry ice.
- Place vial in a 37ºC water bath just long enough to thaw.
- Mix gently and transfer 0.9 mL of cells to a 1.5 mL tube. The remainder of cells should be placed in a control rate freezing jar and placed at -80ºC. Transfer back to the original freezer boxes at -80ºC. Make a note in the MPD Frozen Samples that 1/2 of the sample was used.
- Spin at 600g. x 2 min.
- Pipet off supernatant and discard. Do not use an aspirator because lysed cells may release viscous DNA which is adherent to the cell pellet.
- Dislodge cell pellet by flicking tube and resuspend in 0.2 mL cold PBS.
- DNA: Transfer 0.1 mL to a tube with 0.4 mL of cell lysis buffer and continue with step 7, below.
- Fixed cells: Transfer 20 µL to a tube with 0.5 mL fixative (1:3, HOAc:MeOH). Spot 1 drop onto a glass slide and stain with Hema3 stain. Transfer remainder to a 1.8 mL cryo vial and store at -20ºC. List fixed cells and histo slide in MPD freezer database and Human Tissue Reports.
- RNA: Spin remainder of cells. Discard supernatant. Add 0.6 mL Trizol. See Trizol protocol for details on RNA preparation. Store RNA in 70% EtOH at -20ºC. List RNA samples in MPD freezer database and Human Tissue Reports.
- Place DNA in lysis buffer with proteinase K at 50ºC x 1hr, and mix periodically.
- Add 2 vol. (1 mL) of phenol. Mix gently at 4ºC for 1 hr. to overnight. Transfer aqueous phase to a fresh tube.
- Add 1 vol. (0.5 mL) of CHCl3, mix gently to remove phenol. Transfer aqueous phase to a fresh tube.
- Ethanol precipiptate by adding 2 vol. (1 mL) EtOH, mix and incubate -20ºC for at least 1 hr. Spin at 12,000 rpm to pellet DNA and discard supernatant.
- Wash with 70% EtOH x2. Air dry. Resuspend in 0.1 mL T.E. Store at -80ºC. List DNA samples in MPD freezer database and Human Tissue Reports.