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== Extraction of gDNA of the cell pellet samples from Sergio == * First, I need to test if the column from QIAamp DNA Micro kit still work well since Dinh said that the columns coming with this kit were not stored at 4C more than 1 month, and we 're concerned about the performance of the purification. The cells to be tested are few samples from Sergio which he doesn't want to include in the experiment. '''Cell samples''' 1. HUES2 p38 => code Hues2 2. ASThiPS4F1 p.8 => code AST4F1 3. ASThiPS4F2 p.8 => code AST4F2 '''Follow protocol''' '''Purification of Total DNA from Animal Blood or Cells (Spin-Column Protocol)''' Note: Use the protocol of DNeasy Blood and tissue kit, but use column and buffer come with QIAamp DNA microkit (buffers are the same, but used different volume because the scale) '''Prepare''' 1. Add 25 ml ethanol (96–100%) to the bottle containing 19 ml Buffer AW1 concentrate 2. Add 30 ml ethanol (96–100%) to the bottle containing 13 ml Buffer AW2 concentrate '''Procedure''' 1. Resuspend the pellet in 200 μl PBS. Add 20 μl proteinase K 2. Add 200 μl Buffer AL (without added ethanol). Mix thoroughly by vortexing, and incubate at 56°C for 10 min 3. Add 200 μl ethanol (96–100%) to the sample, and mix thoroughly by vortexing 4. Pipet the mixture from step 3 into the DNeasy Mini spin column placed in a 2 ml collection tube (provided). Centrifuge at 6000 x g (8000 rpm) for 1 min. Discard flow-through and collection tube. 5. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 μl Buffer AW1, and centrifuge for 1 min at 6000 x g (8000 rpm). Discard flow-through and collection tube 6. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 μl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube 7. Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube (not provided), and pipet 100 μl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at 6000 x g (8000 rpm) to elute. Second elute (100ul) '''Concentration by Nanodrop {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Lab ID''' | align="center" style="background:#f0f0f0;"|'''Conc. Elute1 (100ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ug)''' | align="center" style="background:#f0f0f0;"|'''260/280''' | align="center" style="background:#f0f0f0;"|'''Conc. Elute2 (100ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ug)''' | align="center" style="background:#f0f0f0;"|'''Conc. Elute1+2 (200ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ug)''' |- | 1. HUES2 p38 ||HUES2||363.8||36.38||2.04||153.5||15.35||256.6||51.32 |- | 2. ASThiPS4F1 ||4F1||695.7||69.57||2.04||285.9||28.59||486.9||97.38 |- | 3. ASThiPS4F2||4F2||548.3||54.83||1.99||230.1||23.01||390.8||78.16 |} * Since the yields seem to be very high, I wonder there might be some RNA contamination. I ran 5ul of gDNA in E-gel. [[File:ZhangLab_2 2012-05-17 16hr 38min_gDNA-check.jpg| 180px]] Lane1: HUES2 p38 Lane2: ASThiPS4F1 Lane3: ASThiPS4F2 From the image above, the gDNA band was very intact and the intensity was consistent to concentration and no RNA contamination. I then go ahead to extract gDNA for the rest samples.
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