Noi/NOTES/2014-6-9
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DNA extraction of 10 plasma samples and 5 matched pair solid tumor tissue samples from Cancer Center[edit]
- [Link to calendar]
- Part 1: I extract 10 plasma DNA samples from cancer patients received on 2014-06-05
- Part 2: I extract 5 matched pair solid tumor tissue samples from cancer patients received on 2014-05-27
- I assigned new sample codes to be more convenient for sample handling. --> In the future, I may only use the last 3 digit of project ID, so I don't need to rerun sample code and prevent confusion.
Part 1: DNA extraction from plasma samples[edit]
- There are three tubes of plasma for each sample. Dr. Zhang suggested to use 2 tubes for DNA extraction.
- I measured volume of two tube serum during DNA extraction and adjust volume to 600ul with 1X PBS as ChargeSwitch kit required different volume of reagent for different sample volume.
- Note that the measurement could be +-5ul error since serum is viscous and I needed to adjust the pipette volume after aspiration to measure the volume
Project ID # Zhang lab ID Volume 2 tubes PBS to 600ul UCSD-004-04 PC-P_1 560 40 UCSD-004-05 PC-P_2 475 125 UCSD-004-06 PC-P_3 475 125 UCSD-004-07 PC-P_4 330 270 UCSD-004-08 PC-P_5 475 125 UCSD-004-09 PC-P_6 480 120 UCSD-004-10 PC-P_7 480 120 UCSD-004-11 PC-P_8 450 150 UCSD-004-12 PC-P_9 600 0 UCSD-004-13 PC-P_10 440 160
- I followed instruction of ChargeSwith gDNA 1 ml Serum kit as described in [[1]] for 600ul sample volume
Prep
- Eluted 30ul with Elution Buffer
- Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution)
Qubit dsDNA HS assay result[edit]
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Sample conc. (ng/uL) | Volume (ul) | Yields (ng) |
PC-P_1 | 4.73 | ng/mL | 2 | 100 | 0.473 | 30.00 | 14.19 |
PC-P_2 | 8.52 | ng/mL | 2 | 100 | 0.852 | 30.00 | 25.56 |
PC-P_3 | 13.6 | ng/mL | 2 | 100 | 1.360 | 30.00 | 40.80 |
PC-P_4 | 3.4 | ng/mL | 2 | 100 | 0.340 | 30.00 | 10.20 |
PC-P_5 | 8.13 | ng/mL | 2 | 100 | 0.813 | 30.00 | 24.39 |
PC-P_6 | 7.67 | ng/mL | 2 | 100 | 0.767 | 30.00 | 23.01 |
PC-P_7 | 6.44 | ng/mL | 2 | 100 | 0.644 | 30.00 | 19.32 |
PC-P_8 | 5.24 | ng/mL | 2 | 100 | 0.524 | 30.00 | 15.72 |
PC-P_9 | 2.38 | ng/mL | 2 | 100 | 0.238 | 30.00 | 7.14 |
PC-P_10 | 3.7 | ng/mL | 2 | 100 | 0.370 | 30.00 | 11.10 |
Normalize DNA yield to 1mL serum volume[edit]
Sample | Yields (ng) | Plasma volume (ul) | Normalized yield to 1mL (ng) |
PC-P_1 | 14.19 | 560 | 25.34 |
PC-P_2 | 25.56 | 475 | 53.81 |
PC-P_3 | 40.80 | 475 | 85.89 |
PC-P_4 | 10.20 | 330 | 30.91 |
PC-P_5 | 24.39 | 475 | 51.35 |
PC-P_6 | 23.01 | 480 | 47.94 |
PC-P_7 | 19.32 | 480 | 40.25 |
PC-P_8 | 15.72 | 450 | 34.93 |
PC-P_9 | 7.14 | 600 | 11.90 |
PC-P_10 | 11.10 | 440 | 25.23 |
Plasma color (in lysis buffer)[edit]
- Row 1: PC-P_1-5
- Row 2: PC-P_6-10
- File:2014-06-14 PC-P 1-10color.jpg
Part 2: DNA extraction from solid tumor tissue samples[edit]
- The size of tissue samples are varied. Sharmeela noted that they should be ~25mg.
- I used DNeasy Blood and Tissue kit from QIAGEN and followed the instruction of the manufacturer.
Procedures[edit]
- Followed protocol of DNeasy Blood and tissue kit: [[2]]. Page 28.
- I added RNase A to remove RNA contamination
- - Rui suggested that if the tissue is large, I should increase volume of ATL buffer ~3X.
- - I added 180*3ul = 540ul of ATL buffer + 50ul of 10mg/mL RNase A (actually should right before Buffer AL) + 60ul of ProteinaseK
- - Incubate at 56C for ~1h with shaking 900rpm/min and occasionally vigorously vortexing for every 10min.
- I did not use homogenizer to homogenize tissue as it might be difficult to handle and it will generate a lot of bubbles.
- - Anyway, Vortexing and shaking on thermo mixer is good enough to lyse the tissue.
- - After the tissue is completely lysed, I split it into two tube (~330ul each) + added 330ul of Buffer AL
- Eluted 1st time with 200ul AE Buffer
- Eluted 2nd time with 50ul AE Buffer (total each column ~250ul
- Pooled two tubes together --> total volume left ~480ul
- Measure concentration with Qubit dsDNA HS assay using 2ul of extracted DNA in 200ul reaction (100x dilution). Samples PC-T_1 has very high concentration. I repeated measurement and reduced volume to 1ul.
Qubit dsDNA HS assay result[edit]
Sample | Conc. In the Qubit | Unit | uL used | Dilution | Sample conc. (ng/ul) | Volume (ul) | Yield in 480ul (ug) |
PC-T-1 | 413 | ng/mL | 1 | 200 | 82.7 | 480 | 39.70 |
PC-T-2 | 358 | ng/mL | 2 | 100 | 35.8 | 480 | 17.18 |
PC-T-4 | 428 | ng/mL | 2 | 100 | 42.8 | 480 | 20.54 |
PC-T-6 | 152 | ng/mL | 2 | 100 | 15.2 | 480 | 7.30 |
PC-T-7 | 163 | ng/mL | 2 | 100 | 16.3 | 480 | 7.82 |
- I also measured with ND to determined DNA extraction quality and compare measurement by Qubit and ND
Sample | ND conc. (ng/ul) | 260/280 | 260/230 |
PC-T_1 | 89.30 | 1.91 | 2.25 |
PC-T_2 | 40.70 | 1.82 | 1.94 |
PC-T_4 | 44.20 | 1.89 | 2.12 |
PC-T_6 | 15.30 | 2.17 | 2.13 |
PC-T_7 | 16.70 | 1.81 | 1.92 |
- The concentrations from both Qubit and ND are very close this meant that there is very low protein and salt contamination.