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.