Noi/NOTES/2014-4-4

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MONOD test on human plasma/serum[edit]

  • Continued from : [[1]]
  • From last time lab meeting, Dr. Zhang suggested to do capture on serum DNA before continuing to capture with real cancer/tumor samples.
- The sample #2-S-CS I had highest yield last time has ~25ng left after quantification. I extracted more DNA from plasma and serum of sample #3 to increase DNA yield.

ChargeSwitch® gDNA 1 ml Serum kit[edit]

Procedures[edit]

- Add plasma or serum to 1.5mL tube (adjust to total 600ul).
#3-P-CS : plasma volume 560 (pooled 4tubes) + 40ul 1x PBS
#3-S-CS : serum volume 420ul (pooled 3 tubes) + 180ul
- Note: the volume of sample could be up to 1000ul
- Mix 840ul of Lysis Buffer + 60 Proteinase K (420:30, 2 tubes)
- Add 450ul of Lysis Buffer and Proteinase K mix soulution (420ul of Lysis Buffer and 30ul of Proteinase K for 600ul 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
- In 1.5 mL tube, mix 150ul of ChargeSwitch Purification Buffer + 18ul of ChargeSwitch Magnetic beads for 2.1 rxn (315ul buffer + 37.8ul beads)
- Add 168ul 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. )
- Carefully remove the spnt without disturbing the bead pellet
Washing DNA
- Remove sample tube from magnetic rack
- Add 600ul 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
- I dried up the bead for 2 min
Eluting DNA
- Remove sample tube from magnetic rack (there should be no spnt in the tube)
- Add 26ul of ChargeSwitch Elution Buffer to the tube and pipette up & down gently 10x to resuspend magnetic beads
- Wait for 2 min
# 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
- The actual volume left was 28ul. This might be due to the left over washing buffer.

Qubit dsDNA HS assay[edit]

- I used 2ul for assay (100x)
#3-P-CS = 0.246ug/mL or ng/ul, yield = 28ul * 0.246ng/ul = 6.89ng
#3-S-CS = 7.630ug/mL or ng/ul, yield = 28ul * 7.630ng/ul = 213ng

Bisulfite conversion (Zymo Lightning kit)[edit]

- I used 25ul of #3-S-CS for bisulfite conversion (190.75ng). Since the volume exceed 20ul, I adjust Lightning Conversion Reagent accordingly to the input volume.

- Add 162.5ul of Lightning Conversion Reagent to 25ul extracted DNA and mix by pipetting 10X - Follow bisulfite conversion of EZ DNA Methylation-Lightning™ Kit - Elute with 15ul of Elution buffer --> volume after spin is ~14ul

Qubit ssDNA assay[edit]

- I used 1ul for Qubit ssDNA assay (200x dilution)
- bis-#3-S-CS = 7.43ng/ul, yield = 14ul * 7.43ng/ul =104.02
- Recovery rate = (104.02 * 100)/190.75 = 54.53%
- Amount left in 13ul for BSPP capture = 96.59ng