Noi/NOTES/2014-3-11

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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 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.