Noi/NOTES/2014-3-11
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MONOD test on human plasma/serum
- Continued from: [[1]]
Sample labeling
' | Labeling on original tubes | Labeling on working tubes | ||||
Sample ID | Plasma (P) | Serum (S) | Buffy coat (BC) | Plasma (P) | Serum (S) | Buffy coat (BC) |
UCSD004.1 | UCSD004#1-P-Q | UCSD004#1-S-Q | UCSD004#1-P-Q | #1-P-Q | #1-S-Q | #1-P-Q |
UCSD004#1-P-Nu | UCSD004#1-S-Nu | UCSD004#1-P-Nu | #1-P-Nu | #1-S-Nu | #1-P-Nu | |
UCSD004#1-P-CS | UCSD004#1-S-CS | UCSD004#1-P-CS | #1-P-CS | #1-S-CS | #1-P-CS | |
UCSD004#1-P-Z | UCSD004#1-S-Z | UCSD004#1-P-Z | #1-P-Z | #1-S-Z | #1-P-Z | |
UCSD004.2 | UCSD004#2-P-Q | UCSD004#2-S-Q | UCSD004#2-P-Q | #2-P-Q | #2-S-Q | #2-P-Q |
UCSD004#2-P-Nu | UCSD004#2-S-Nu | UCSD004#2-P-Nu | #2-P-Nu | #2-S-Nu | #2-P-Nu | |
UCSD004#2-P-CS | UCSD004#2-S-CS | UCSD004#2-P-CS | #2-P-CS | #2-S-CS | #2-P-CS | |
UCSD004#2-P-Z | UCSD004#2-S-Z | UCSD004#2-P-Z | #2-P-Z | #2-S-Z | #2-P-Z |
Order in sample IDs/codes | Description |
1 | Project ID |
2 | # of sample (1,2,3,…,N) |
3 | Type of blood fraction |
4 | Extracted by kit |
P | Plasma |
S | Serum |
BC | Buffy coat |
Q | Extracted by QIAGEN kit |
Nu | Extract by nucleospin column |
CS | Extracted by ChargeSwitch kit |
Z | Extracted by Zymo kit |
Serum/Plasma DNA extraction
- - For column-based method, I will use 200ul of plasma/serum/buffy coat
1. QIAamp DNA Blood Mini Kit
- - Follow protocol described in [[2]]
- - Resuspend lyophilized QIAGEN Protease with 1.2 ml protease solvent.
Procedures
- Preparing lysate
- - Pipet 20 µl of QIAGEN Protease (or proteinase K) into the bottom of a 1.5 ml tube
- - Add 200 µl sample to the tube. If the volume of sample less than 200ul, adjust volume with PBS
- - Note: All samples were provided at different volumes, I used different volumes for DNA extraction. At the end, the yields by each method will be compared with normalized sample volume.
Samples Volume (ul) 1X PBS (ul) #1-P-Q 100.00 100.00 #1-S-Q 200.00 0.00 #1-BC-Q 100.00 100.00 #2-P-Q 150.00 50.00 #2-S-Q 200.00 0.00 #2-BC-Q 100.00 100.00
- - Add 200 µl of Buffer AL to the sample. Mix by pulse-vortexing for 15 s
- - Incubate at 56°C for 10 min
- Binding DNA
- - Briefly spin down the tube before adding 200ul 100% EtOH. Mix again by pulse-vortexing for 15 s and briefly spin down
- - Transfer spnt to QIAamp Mini spin column & centrifuge at 6,000xg for 1 min
- Washing DNA
- - Transfer QIAamp Mini spin column in a clean 2 ml collection tube
- - Add 500 µl of Buffer AW1 & centrifuge at 6,000xg for 1 min
- - Transfer QIAamp Mini spin column in a clean 2 ml collection tube
- - Add 500 µl of Buffer AW2 & centrifuge at 20,000xg for 3min
- Eluting DNA
- - Transfer the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at 20,000xg for 1 min (full speed)
- - Place the QIAamp Mini spin column in a clean 1.5 ml tube & add appropriate volume of Buffer AE or distilled water (manual recommends 200ul). --> I eluted with 26ul Buffer AE
- - Incubate at RT for 5 min & centrifuge at 6,000xg for 1 min
2. NucleoSpin® Plasma XS kit
- - Follow protocol described in [[3]]
- - Add 1.35mL of Proteinase Buffer (PB) to 30mg of lyophilized Proteinase K for 50 prep kit.
Procedures
- # To remove residual cells, centrifuge plasma/serum at >11,000xg for 3 min
- Preparing lysate
- - Add 200ul of plasma/serum/buffy coat to 1.5mL tube.
Samples Volume (ul) 1X PBS (ul) #1-P-Nu 100.00 100.00 #1-S-Nu 200.00 0.00 #1-BC-Nu 100.00 100.00 #2-P-Nu 200.00 0.00 #2-S-Nu 150.00 50.00 #2-BC-Nu 100.00 100.00
- - Add 20ul of proteinase K to plasma/serum sample
- - Mix and incubate at 37C for 10min
- Binding DNA
- - Add 1.5 volume (300ul) of Buffer BB (keep sample:binding buffer ratio 1:1.5 v/v)
- - Invert the tube 3x and vortex for 3s. Spin down the tube briefly.
- - Load 600ul of the mixture to a Nucleospin Plasma XS column
- - Centrifuge at 2,000xg for 30s. Centrifuge at higher speed at 11,000xg for further 5s.
- Washing DNA
- - Transfer Nucleospin column to a new collecting tube
- - Add 500ul of buffer WB onto the column. Centrifuge at 11,000xg for 30s.
- - Place column into new collecting tube
- - Add 250ul of buffer WB to the column. Centrifuge at 11,000xg for 3min
- Eluting DNA
- - Place column to 1.5mL tube
- - Add 26ul of Elution buffer to the column (range 5-30ul)
- - Centrifuge at 11,000xg for 30s
- - Eluted DNA from buffy coat samples was red because erythrocytes were carried over with buffy coat layer either in sample #1 or #2.
- - Incubate eluted DNA with open lid at 90C for 8 min to remove residual ethanol --> I skipped this step by drying the column for 3 min before eluting DNA.
- - For bead-based method, I will split samples for two processes. One for regular DNA extraction and one will go to bisulfite treatment after DNA binding to beads without washing and elution.
- - The volume of sample was not exactly 200ul (plasma and buffy coat ~100ul for each, serum ~200ul), so I used different volume for DNA extraction (50, 100 or 200ul)
3. ChargeSwitch® gDNA 1 ml Serum kit
Procedures
- - Add plasma and sample to 1.5mL tube (adjust to total 200ul).
- - Note: the volume of sample could be up to 1000ul
Samples Volume (ul) 1X PBS (ul) #1-P-CS 50.00 150.00 #1-S-CS 100.00 100.00 #1-BC-CS 50.00 150.00 #2-P-CS 50.00 150.00 #2-S-CS 100.00 100.00 #2-BC-CS 50.00 150.00
- - Mix 910ul of Lysis Buffer + 195 Proteinase K (140:30, 2 tubes)
- - Add 170ul of Lysis Buffer and Proteinase K mix soulution (140ul of Lysis Buffer and 30ul of Proteinase K for 200ul sample)
- - Pipette up & down 5x to mix
- - Incubate at RT for 20min to lyse the sample.
- Binding DNA
- - Vortex the tube containing ChargeSwitch Magnetic beads
- - Mix 50ul of ChargeSwitch Purification Buffer + 6ul of ChargeSwitch Magnetic beads for 13 rxn (650ul buffer + 78ul beads)
- - Add 56ul of ChargeSwitch Purification Buffer and ChargeSwitch Magnetic beads mix to the digested sample
- - Pipette up & down 5x to mix (use p1000 pipette, avoid generating bubbles)
- - Incubate at RT for 2 min to allow DNA bind to magnetic beads
- - Place the sample tube in the MagnaRack for 3 min or until the bead forming a tight pellet. (I may use magnetic rack for 1.5mL tube available in the lab)
- - Carefully remove the spnt without disturbing the bead pellet
- Washing DNA
- - Remove sample tube from magnetic rack
- - Add 200ul of ChargeSwitch Wash Buffer to the tube and pipette up & down 5x gently & avoid bubbles
- - Place the sample tube in the MagnaRack for 2 min or until the bead forming a tight pellet
- - Carefully remove the spnt without disturbing the bead pellet
- - Repeat washing step
- Eluting DNA
- - Remove sample tube from magnetic rack (there should be no spnt in the tube)
- - Add 25ul of ChargeSwitch Elution Buffer to the tube and pipette up & down gently 10x to resuspend magnetic beads
- # Do not use H2O for elution
- - Place the sample tube in the MagnaRack for 1 min or until the bead forming a tight pellet
- - Carefully transfer eluted DNA to 1.5mL tube
For bisulfite treatment
- - After DNA binding step prior to washing step, I resuspend the beads with 20ul of ChargeSwitch Elution Buffer
- - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X
- - Transfer solution to 200ul tube
- - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit
- - Elute with 17ul of Elution buffer --> volume after spin is ~16ul
- - Used 3ul for Qubit ssDNA assay
- - Note: This kit was performed after Zymobeads. I saw that 2ul for assay was not enough to be detected for some samples, so I increase the sample volume from 2ul to 3ul.
- - I will calculate total amount in 16ul --> normalize yield to 100ul input sample
Quant-iT ssDNA
Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. Unit Yield in 16ul (ng) #1-P-CS <1ng/mL NA #1-S-CS <1ng/mL NA #1-BC-CS 1.2 ng/mL 3 66.7 79.00 pg/ul 1.26 #2-P-CS <1ng/mL NA #2-S-CS 3.1 ng/mL 3 66.7 210.00 pg/ul 3.36 #2-BC-CS 64.6 ng/mL 3 66.7 4.31 ng/ul 68.96
Normalized to 100ul sample input
Sample Volume used (ul) Yields (ng) Yields in 100ul (ng) #1-P-CS 50 NA #1-S-CS 100 NA #1-BC-CS 50 1.26 2.53 #2-P-CS 50 NA #2-S-CS 100 3.36 3.36 #2-BC-CS 50 68.96 137.92
4. ZymoBead™ Genomic DNA kit
Procedures
- -Before starting, add 250ul of beta-mercaptoethanol to 50mL of Genomic Lysis Buffer to a final dilution of 0.5% (v/v). Vortex ZymoBead to resuspend
- Preparing lysate and binding DNA
- - Add 200ul of Genomic Lysis Buffer to 50ul samples in 1.5mL tube
- - Add 10ul ZymoBeads. Mix by inversion
- - Incubate at RT for 5 min
- - Centrifuge at 1,500xg for 1 min
- - Carefully remove the spnt without disturbing the bead pellet
- - Add 200ul of Genomic Lysis Buffer to the ZymoBeads
- Resuspend the beads by pipetting up & down
- Centrifuge at 1,500xg for 1 min
- - Carefully remove the spnt without disturbing the bead pellet
- Washing DNA
- - Add 200ul of DNA Pre-Wash Buffer to the ZymoBeads.
- - Resuspend the pellet, transfer to a new tube, centrifuge at 1,500xg for 1min
- - Carefully remove the spnt without disturbing the bead pellet
- Add 500ul of g-DNA Wash Buffer to the ZymoBeads.
- - Resuspend the pellet and centrifuge at 1,500xg for 1 min.
- - Carefully remove the spnt without disturbing the bead pellet
- Re-centrifuge briefly and remove the residual wash buffer
- Eluting DNA
- - Add 35ul of Elution Buffer and resuspend the pellet by pipetting up & down.
- - Centrifuge at 10,000xg for 1 min
- - Collect the spnt to the 1.5mL tube tube.
For bisulfite treatment
- - After DNA binding step prior to pre-washing step, I resuspend the beads with 20ul of Elution Buffer
- - Add 130ul of Lightning Conversion Reagent and mix by pipetting 10X
- - Transfer solution to 200ul tube
- - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit
- - Elute with 17ul of Elution buffer --> volume after spin is ~16ul
- - Used 2ul for Qubit ssDNA assay
- - I will calculate total amount in 16ul --> normalize yield to 100ul input sample
Quant-iT ssDNA
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Sample Conc. | Unit | Yield in 16ul (ng) |
#1-P-Z | <1ng/mL | ||||||
#1-S-Z | 1.3 | ng/mL | 2 | 100 | 130.00 | pg/ul | 1.82 |
#1-BC-Z | 12.6 | ng/mL | 2 | 100 | 1.26 | ng/ul | 17.64 |
#2-P-Z | 1.5 | ng/mL | 2 | 100 | 150.00 | pg/ul | 2.10 |
#2-S-Z | 3.1 | ng/mL | 2 | 100 | 310.00 | pg/ul | 4.34 |
#2-BC-Z | 38 | ng/mL | 2 | 100 | 3.80 | ng/ul | 53.20 |
Normalized to 100ul sample input
Sample | Volume used (ul) | Yields (ng) | Yields in 100ul (ng) |
#1-P-Z | 50 | NA | NA |
#1-S-Z | 50 | 1.82 | 3.64 |
#1-BC-Z | 50 | 17.64 | 35.28 |
#2-P-Z | 50 | 2.10 | 4.20 |
#2-S-Z | 50 | 4.34 | 8.68 |
#2-BC-Z | 50 | 53.20 | 106.40 |
- - For comparison of the four kits in DNA extraction efficiency, I am waiting for Richard's help for Bioanalyzer HS DNA measurement of 24 sample before continue to bisulfite conversion step.
- - Richard helped me to do Bioanalyzer analysis. There were some samples that I suspected that the labeling may be wrong (between 2-P-Nu and 2-S-Nu).
Bioanalyzer analysis results
- - I can not use microplate reader in Dr. Sah's lab now, so I will do Qubit dsDNA HS assay to quantify concentration and confirm the intensity profile by bioanalyzer analysis before continue to bisulfite conversion.
Qubit dsDNA HS assay results
Raw data befor normalization
- Sample #1
Sample Conc. in the Qubit Unit uL used Dilution Sample Con. Unit Eluted volume (ul) Amount (ng) #1-P-Q <.5ng/mL NA 24 NA #1-P-Nu <.5ng/mL NA 24 NA #1-P-CS <.5ng/mL NA 23 NA #1-P-Z <.5ng/mL Na 33 NA #1-S-Q 2.59 ng/mL 2 100 0.26 ug/mL 24.00 6.22 #1-S-Nu 2.23 ng/mL 2 100 0.22 ug/mL 24.00 5.35 #1-S-CS 3.78 ng/mL 2 100 0.38 ug/mL 23.00 8.69 #1-S-Z <.5ng/mL NA 33.00 NA #1-BC-Q 530 ng/mL 1 200 110.00 ug/mL 24.00 2,640.00 #1-BC-Nu 67.7 ng/mL 2 100 6.77 ug/mL 24.00 162.48 #1-BC-CS 12.2 ng/mL 2 100 1.22 ug/mL 23.00 28.06 #1-BC-Z 94 ng/mL 2 100 9.40 ug/mL 33.00 310.20
- Sample #2
Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. Unit Eluted volume (ul) Amount (ng) #2-P-Q <.5ng/mL NA 24.00 NA #2-P-Nu 14.8 ng/mL 2 100 1.48 ug/mL 24.00 35.52 #2-P-CS 0.85 ng/mL 2 100 0.09 ug/mL 23.00 1.96 #2-P-Z <.5ng/mL NA 33.00 NA #2-S-Q 15.7 ng/mL 2 100 1.57 ug/mL 24.00 37.68 #2-S-Nu 0.52 ng/mL 2 100 0.05 ug/mL 24.00 1.25 #2-S-CS 18.1 ng/mL 2 100 1.81 ug/mL 23.00 41.63 #2-S-Z 3.58 ng/mL 2 100 0.36 ug/mL 33.00 11.81 #2-BC-Q 143 ng/mL 1 of 10x dilution 2000 286.00 ug/mL 24.00 6,864.00 #2-BC-Nu 474 ng/mL 2 100 47.40 ug/mL 24.00 1,137.60 #2-BC-CS 19.5 ng/mL 1 200 3.90 ug/mL 23.00 89.70 #2-BC-Z 102 ng/mL 1 200 20.40 ug/mL 33.00 673.20