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 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.