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 Meaning 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
- - 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 25ul 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 240ul of plasma/serum to 1.5mL tube. --> I will add 200ul
- - Add 20ul of proteinase K to plasma/serum sample
- - Mix and incubate at 37C for 10min
- Binding DNA
- - Add 1.5 volume (360ul) of Buffer BB (keep sample:binding buffer ration 1:1.5 v/v) --> should be 300ul of Buffer BB
- - 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 25ul of Elution buffer to the column (range 5-30ul)
- - Centrifuge at 11,000xg for 30s
- - 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.