Sam:LabNotes/Microbiome/2009-3-12 exp2

From ZhangLabWiki
Jump to navigation Jump to search

Large scale MDA on diluted whole cells using - L4N6 primer plus Tre[edit]

Objective[edit]

  • The L4N6 (locked nucleic acid primer) plus Tre (1.2 M) showed the best result of amplifying curves (on 3-10-09' exp) which can differenciate one single cell and background the most. The current experiment is designed to capture the diluted whole cell using the same set up.

Sample & Material[edit]

  • Primer:L4N6 primer
  • Cell: GM18506 cell line, harvested, washed by UV-treated PBS and coundted to 200,000 cell/mL (200 cell/uL)
    • Dilute to 10 cell/uL, 5 cell/uL, 0.5 cell/uL using UV-treated PBS
  Dilute factor     original    x 1/2   x 1/10   x 1/2    x 1/10
  conc.(cell/uL)      200/uL   100/uL    10/uL    5/uL    0.5/uL
  Transfering(uL)           50        10       50      10                     
  UVed-PBS(uL)                   50       90       50      90


  • Pos. control: human gDNA diluted to 3 ng/uL
    • Measure the original (commercial human gDNA) using Nanodrop -> 309ng/uL
  Dilute factor    original     x 1/100       x 1/10     x 1/10    x 1/10
  conc.            309ng/uL      3ng/uL     300pg/uL    30pg/uL    3pg/uL
  Transfering(uL)            1           10          10         10                     
  UVed-PBS(uL)                     99           90         90         90


  • Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
    • Prepare 5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH.
    • Mix 20 ul 5M KOH, 5 ul 0.5M EDTA, 25 ul 1M DTT(Qiagen), 200 ul nuclease free H2O (Note: this is new formula)
    • Mix 10ul ALS buffer with 10ul NS buffer, check pH with a pH paper, adjust ALS buffer with KOH or H2O if pH is not within 7-8
  0.093 g (KOH=56.11) = 0.001657 mole;   0.001657/5 x 10^6 =331.49 uL --- H2O needed


Reaction arrangement[edit]

  • Left(A) to Right(H)
    • Row1: 3ng, 300pg, 30pg, 3pg x 3, H2O x 2
    • Row2: 0.5 cell x 8
    • Row3: 0.5 cell x 8
    • Row4: 0.5 cell x 6, 10 cell, 5 cell
  • Total = 32 rxn -> Prepare (8+1)x4 =36 rxns for master mix


Procedures[edit]

  • Thaw the reagents on ice and UV-treated H2O, 1XPBS and equipment for 10 min
  • The the cool block out from frezor and leave in room tempterature for at least 10 min (To prevent the over-cold problem)
  • Dilute the gDNA in UV-treated H2O into 3ng/uL, 300pg/uL, 30pg/uL, 3pg/uL
  • Prepare ALS buffer and test the pH (use old NS buffer is ok)
  • Transfer 250 uL(or enough amount) of ALS, NS, and dH2O into PCR tubes and UV-treated for 10 min (using cross linking machine)
  • At the same time, dilute the cell with UV-treated PBS into 10 cell/uL, 5 cell/uL and 0.5 cell/uL
  • Prepare the 2X SYBR (2uL of 50X SYBR + 48 uL H2O)
  • Prepare MDA master mix (in a 0.6-mL tube)and leave on ice
                                1 rxn      x 36 rxn
  -------------------------------------------------
  H2O                            6.2 uL   223.2 uL
  1mM L4N6 primer                1.0 uL    36.0 uL
  10x RepliPhi phi-29 buffer     2.0 uL    72.0 uL
  25mM dNTP                      0.8 uL    28.8 uL 
  2X SYBR Green I                1.0 uL    36.0 uL
  RepliPhi Phi-29 (100U/ul)      1.0 uL    36.0 uL
  -------------------------------------------------
                                          432.0 uL   (432/36=12)


  • Prepare cell lysates:
    • Trasfer 1 uL ALS buffer into each of 32 PCR tubes on the cool-block
    • Transfer 1 uL template (Pos, Neg, diluted whole cell) into each of 32 PCR tubes as prevous indication, mix with pipetting in each transferring.
    • Incubate for 5 min on cool block
    • Transfer 1 uL NS buffer into each of 32 PCR tubes, mix with pipetting.
  • Transfer 5 uL of Tre(1.2M) into each of 32 PCR tubes, mix with pipetting.
  • Transfer 12 uL MDA master mix to each of 32 PCR tubes. Mix with pipetting.
  • Perfomr realtime PCR in Chromo4 with the new program
  line 1. Incubate at 30C for 6 min
  line 2. Plate read
  line 3. Goto line 1 for 99 more times
  line 4. Incubate at 30C for 6 min
  line 5. Plate read
  line 6. Goto line 4 for 99 more times
  line 7. Incubate at 85C for 3 min
  line 8. Incubate at 4C forever
  Note: Tre treatment will delay the reaction time, so we put 200 cycles in total. (The maximum setting is 99 cycles for the program)


  • Stop the program (when the reaction reach the signal saturation) and pick up the samples
  • Inactivate the samples at 85C for 3 min using thermocycler.


Results[edit]

  File:Sam031209-L4N6 Tre.bmp
  Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction