Sam:LabNotes/Human/2009-4-17

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Human MDA on L4N9, L4N8, L4N7 and N6 with Tre[edit]

Objective[edit]

  • Exp. Design (total = 32 rxns)
Row A: Primer L4N9   30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3)
Row B: Primer L4N8   30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3)
Row C: Primer L4N7   30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3)
Row D: Primer  N6    30 pg (x2), 3 pg (x2), 3 fg (x1), H2O (x3)


Preparation:[edit]

  • Thaw all the reagents needed for MDA
    • 10X buffer (from kit)
    • Ambion RT-PCR H2O
    • 1 mM DTT (Sigma)
    • dNTP (from kit)
    • N6 (1mM)
    • pre-diluted gDNA standard (30pg, 3pg, 3 fg)
  • Prepare fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
    • 5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH. Ex. 0.093 g (KOH=56.11) = 0.001657 mole; 0.001657/5 x 10^6 =331.49 uL --- H2O needed
    • Mix 200ul nuclease free H2O, 25ul 1M DTT(Sigma), 20ul 5M KOH, 5ul 0.5M EDTA
    • 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
  • Extra-UV treatment for buffers and working solution-UV 15 min with the cap open
    • RT-PCR grade H2O (Ambion)- 20 uL x 1 PCR tubes - for Blank
    • ALS buffer - 20 uL x 4 PCR tubes (for 4 rows)
    • NS buffer - 20 uL x 4 PCR tubes (for 4 rows)
  • Set up Bio-Rad realtime PCR programing:
         Line 1: 30 C, 6 min
         Line 2: Plate read
         Line 3: Goto line 1 for additional 99 times
         Line 4: 30 C, 6 min
         Line 5: Plate read
         Line 6: Goto line 1 for additional 99 times
         Line 7: 85 C, 3 min (inactivation)
         Line 8: 4 C, forever
         Line 9: End
  • Prepare 2X SYBR (2 uL of 50X SYBR + 48 uL H2O)
  • Prepare No-dNTP-MDA master mix (in a 1.5-mL tube)
                                   1 rxn           9 rxns  X 4          
      -------------------------------------------------------------
      RT-PCR grade H2O               5.2           46.8 uL           
      10x RepliPhi phi-29 buffer     2.0           18.0 uL              
      1mM  primer                    1.0            9.0 uL  - L4N9, L4N8,  L4N7, N6
      2X SYBR Green I                1.0            9.0 uL                  
      RepliPhi Phi-29 (100U/ul)      1.0            9.0 uL                  
     --------------------------------------------------------------
                                    10.2           91.8 uL  (91.8/9=10.2)                
                                                      
     25mM dNTP                       0.8 uL         7.2 uL
     --- add dNTP when reactions are ready to set up
  
  • Warm by the cool rack (from -20) at RT for 10 min
  • Use a p200 pipettor to mix and pool the UVed buffer and H2O together
  • Set up PCR tube strips on the rack at RT. Add 1.5 uL ALS into the tube.
  • Transfer 1 uL of template into each of PCR tubes and mix by gently pipetting (4 times). Incubate at RT for 5 min (This step is still done at RT)
  • Add 1.5 uL NS into the tube and mix by pipetting 4 times
  • Add 5.0 uL Tre into the tube and mix by pipetting 4 times

!!! Add the dNTP into master mix !!!

  • After add dNTP in phi29 master mix, transfer 11 uL master mix into each of tubes. Cap and label the tube. Mix by vortexing and quick spinning down.
  • Place the reaction in Bio-Rad realtime PCR machine and start the reaction program (using 20-hr incubation setting).
  • Inactivate all amplicons for 85C 3min

Results[edit]

  File:041709 L4N9-tre.jpg File:041709 L4N8-tre.jpg 

  File:041709 L4N7-tre.jpg File:041709 N6-tre.jpg

Discussion[edit]

  • Performance (based on seperation between 30pg and NTC)
    • L4N7>L4N8>L4N9>N6
  • Although a whole new phi29 kit (enzyme + buffer) was used in this experiment, a 3fg-level contamination was found in most reactions. It is possible the contamination might exit in 50X SYBR (or primers ?)