Noi/NOTES/2014-5-31: Difference between revisions
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* 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. | * 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. | ||
** I have repeated measuring conc. of PC-S-3 and PC-S-4 and the conc. are 17.2 and 14.7 ng/ul, respectively. These numbers are close to previous measurement. I will use previous numbers as they were measure in the same time of other samples. | |||
==== Normalize DNA yield to 1mL serum volume ==== | ==== Normalize DNA yield to 1mL serum volume ==== | ||
{| {{table}} class = wikitable | {| {{table}} class = wikitable |
Revision as of 23:42, 1 June 2014
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 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
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.
- I have repeated measuring conc. of PC-S-3 and PC-S-4 and the conc. are 17.2 and 14.7 ng/ul, respectively. These numbers are close to previous measurement. I will use previous numbers as they were measure in the same time of other samples.
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. This is still higher than the amount of some non-bisulfite converted samples from this batch