Noi/NOTES/2014-3-11: Difference between revisions

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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.
: - 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 result ===
: - 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.

Revision as of 04:14, 14 March 2014

MONOD test on human plasma/serum

[Link to calendar]

  • 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 50ul 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 result

- 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.