Noi/NOTES/2014-5-31

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DNA extraction of 10 serum samples from Cancer Center

  • [Link to calendar]
  • I extract 10 serum samples from cancer patients received on 2014-05-27 [[1]]
  • There are two tubes of serum, Dr. Zhang suggested me to extract DNA on one tube first to see the result from tube and save the second tube in case there is something wrong with the first experiment
  • I assigned new sample codes to be more convenient for sample handling.
  • I measured volume of the first tube serum during DNA extraction and adjust volume to 400ul with 1X PBS as ChargeSwitch kit required different volume of reagent for different sample volume.
Project ID # Zhang lab ID Volume tube_1 PBS to 400ul
UCSD-004-04 PC-S_1 300 100
UCSD-004-05 PC-S_2 305 95
UCSD-004-06 PC-S_3 165 235
UCSD-004-07 PC-S_4 250 150
UCSD-004-08 PC-S_5 315 85
UCSD-004-09 PC-S_6 315 85
UCSD-004-10 PC-S_7 300 100
UCSD-004-11 PC-S_8 180 220
UCSD-004-12 PC-S_9 250 150
UCSD-004-13 PC-S_10 250 150
  • PC = pancreatic cancer
  • S = serum smaple
  • I followed instruction of ChargeSwith gDNA 1 ml Serum kit as described in [[2]] for 400ul sample volume

Prep

Components 1x 10.5x
Lysis Buffer 280 2,940
Proteinase K 30 315
- Aliquot 310ul
Components 1x 10.5x
Purification Buffer (N7) 100 1,050
Magnetic beads 12 126
- Aliquot 112ul
  • Eluted 32ul 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

Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. (ng/ul) Volume Yield (ng)
PC-S_1 8.79 ng/mL 2 100 0.879 30 26.37
PC-S_2 16.6 ng/mL 2 100 1.660 30 49.80
PC-S_3 214 ng/mL 2 100 21.400 30 642.00
PC-S_4 8.5 ng/mL 2 100 0.850 30 25.50
PC-S_5 15.8 ng/mL 2 100 1.580 30 47.40
PC-S_6 175 ng/mL 2 100 17.500 30 525.00
PC-S_7 18 ng/mL 2 100 1.800 30 54.00
PC-S_8 9.53 ng/mL 2 100 0.953 30 28.59
PC-S_9 57.2 ng/mL 2 100 5.720 30 171.60
PC-S_10 17.1 ng/mL 2 100 1.710 30 51.30
  • I may need to repeat sample PC-S_3 andPC-S_6 as the concentration is very high. To save DNA, I will use 1ul for the assay.

Normalize DNA yield to 1mL serum volume

Sample Yield (ng) Serum volume (ul) Normalize yield to 1mL (ng)
PC-S_1 26.37 300 87.90
PC-S_2 49.80 305 163.28
PC-S_3 642.00 165 3,890.91
PC-S_4 25.50 250 102.00
PC-S_5 47.40 315 150.48
PC-S_6 525.00 315 1,666.67
PC-S_7 54.00 300 180.00
PC-S_8 28.59 180 158.83
PC-S_9 171.60 250 686.40
PC-S_10 51.30 250 205.20
  • Some samples have the yield lower than 50ng. I am afraid that this amount may not be enough for BSPP capture. After bisulfite conversion the yield might be only 10ng or lower.
  • I need to repeat V1N3 capture to confirm the minimum amount of BIS-DNA required for capture
  • For current experiment, the minimum is 50ng that is still higher than the amount of some sample from this batch