Noi/NOTES/2012-5-17

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Extraction of gDNA of the cell pellet samples from Sergio[edit]

  • 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 
Sample IDs Lab ID Conc. Elute1 (100ul) Yields (ug) 260/280 Conc. Elute2 (100ul) Yields (ug) Conc. Elute1+2 (200ul) 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

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.

April 15, 2012, Cell pellet samples from Sergio to Dinh[edit]

No. Sample ID Lab ID
hESC lines:
1 HUES2 p41 Ap15-1
hiPSC lines:
2 ASThiPS4F1 p12 Ap15-2
3 ASThiPS4F2 p12 Ap15-3
Differentiate experiments:
4 HUES6 p39 Control TeSR Ap15-4
5 HUES6 p39 ACTIVIN Ap15-5
6 HUES6 p39 BMP4 Ap15-6
7 HUES9 p39 Control TeSR Ap15-7
8 HUES9 p39 ACTIVIN Ap15-8
9 HUES9 p39 BMP4 Ap15-9
10 HUES3 p36 Control TeSR Ap15-10
11 HUES3 p36 ACTIVIN Ap15-11
12 HUES3 p36 BMP4 Ap15-12
13 H1 p54 Control TeSR Ap15-13
14 H1 p54 ACTIVIN Ap15-14
15 ASThiPS4F5 p16 Control TeSR Ap15-15
16 ASThiPS4F5 p16 ACTIVIN Ap15-16
17 ASThiPS4F4 p36 Control TeSR Ap15-17
18 ASThiPS4F4 p36 ACTIVIN Ap15-18
19 ASThiPS4F4 p36 BMP4 Ap15-19

May 15, 2012, Cell pellet samples from Sergio to Dinh[edit]

No. Sample ID Lab ID
hESC lines:
1 H1 p48 Ma15-2
2 H9 p42 Ma15-3
3 HUES3 p33 Ma15-4
4 HUES6 p26 Ma15-5
5 HUES8 p35 Ma15-6
6 HUES9 p36 Ma15-7
iPSC lines:
7 ASThiPS4F3 p24 (derived from Astrocytes) Ma15-8
8 ASThiPS4F4 p16 (derived from Astrocytes) Ma15-9
9 FiPS4F2 p32 (derived from IMR90 fibroblasts) Ma15-10
10 FiPS4F5 p29 (derived from IMR90 fibroblasts) Ma15-11
11 KiPS4FA p54 (derived from keratinocytes) Ma15-12
12 Huv-iPS4F6 p23 (derived from HUVECs) Ma15-13
Somatic lines:
13 IMR90 p6 Ma15-14
14 Keratinocytes p3 Ma15-15
Differentiation experiments:
15 H1 p47 Control Ma15-16
16 H1 p47 BMP-4 Ma15-17
17 ASThiPS4F5 p12 Control Ma15-18
18 ASThiPS4F5 p12 BMP-4 Ma15-19
19 Huv-iPS4F6 p23 Control Ma15-20
20 Huv-iPS4F6 p23 BMP-4 Ma15-21
21 Huv-iPS4F6 p23 ACTIVIN Ma15-1
Three samples below will not be captured with this batch
HUES2 p38
ASThiPS4F1 p8 (derived from Astrocytes)
ASThiPS4F2 p8 (derived from Astrocytes)


  • gDNA extraction and elute with 250 buffer AE,(except sample Ap15-16 500ul)
No. Sample ID Lab ID Conc. (ng/ul) Yields (ug) in 250ul
1 HUES2 p41 Ap15-1 110.0 27.5
2 ASThiPS4F1 p12 Ap15-2 139.1 34.8
3 ASThiPS4F2 p12 Ap15-3 145.1 36.3
4 HUES6 p39 Control TeSR Ap15-4 295.5 73.9
5 HUES6 p39 ACTIVIN Ap15-5 128.1 32.0
6 HUES6 p39 BMP-4 Ap15-6 153.9 38.5
7 HUES9 p39 Control TeSR Ap15-7 183.5 45.9
8 HUES9 p39 ACTIVIN Ap15-8 149.3 37.3
9 HUES9 p39 BMP-4 Ap15-9 111.2 27.8
10 HUES3 p36 Control TeSR Ap15-10 153.5 38.4
11 HUES3 p36 ACTIVIN Ap15-11 87.5 21.9
12 HUES3 p36 BMP-4 Ap15-12 109.7 27.4
13 H1 p54 Control TeSR Ap15-13 191.3 47.8
14 H1 p54 ACTIVIN Ap15-14 108.7 27.2
15 ASThiPS4F5 p16 Control TeSR Ap15-15 134.2 33.6
*16 ASThiPS4F5 p16 ACTIVIN Ap15-16 50.2 25.1
17 ASThiPS4F4 p36 Control TeSR Ap15-17 183.0 45.8
18 ASThiPS4F4 p36 ACTIVIN Ap15-18 82.6 20.7
19 ASThiPS4F4 p36 BMP-4 Ap15-19 263.4 65.9


  • gDNA extraction and elute with 200 buffer AE,(except sample Ma15-14 150ul)
  • Note: This batch I eluted with smaller volume (200ul except Ma1-14 which had very little of the cell pellet) of AE buffer because I was afraid that the concentration of some samples will be lower than 50ng/ul
No. Sample ID Lab ID Conc. (ng/ul) Yields (ug) in 200ul
1 H1 p48 Ma15-2 215.2 43.0
2 H9 p47 Ma15-3 252.0 50.4
3 HUES3 p33 Ma15-4 226.1 45.2
4 HUES6 p26 Ma15-5 238.7 47.7
5 HUES8 p35 Ma15-6 83.4 16.7
6 HUES9 p36 Ma15-7 236.8 47.4
7 ASThiPS4F3 p24 (derived from Astrocytes) Ma15-8 175.9 35.2
8 ASThiPS4F4 p16 (derived from Astrocytes) Ma15-9 164.3 32.9
9 FiPS4F2 p32 (derived from IMR90 fibroblasts) Ma15-10 205.9 41.2
10 FiPS4F5 p29 (derived from IMR90 fibroblasts) Ma15-11 97.6 19.5
11 KiPS4FA p54 (derived from keratinocytes) Ma15-12 129.5 25.9
12 Huv-iPS4F6 p23 (derived from HUVECs) Ma15-13 263.1 52.6
*13 IMR90 p6 (eluted with 150ul AE buffer) Ma15-14 76.1 11.4
14 Keratinocytes p3 Ma15-15 138.7 27.7
15 H1 p47 Control Ma15-16 216.4 43.3
16 H1 p47 BMP-4 Ma15-17 137.6 27.5
17 ASThiPS4F5 p12 Control Ma15-18 224.6 44.9
18 ASThiPS4F5 p12 BMP-4 Ma15-19 111.7 22.3
19 Huv-iPS4F6 p23 Control Ma15-20 289.9 58.0
20 Huv-iPS4F6 p23 BMP-4 Ma15-21 172.9 34.6
21 Huv-iPS4F6 p23 ACTIVIN Ma15-1 86.7 17.3