AML Sample genomic DNA Preparation

互联网2013-09-06

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<big>Solutions PBS 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 Trizol reagent 75% EtOH made with RNAse free H2 O 100% EtOH Phenol: Buffered to pH=8 with Tris. Chloroform: Stabilized with isoamyl alcohol. T.E.: 10 mM Tris pH8, 1 mM EDTA. Note 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. Procedure </big> <big> </big>

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.