Sam:LabNotes/Microbiome-new/2009-2-20

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E. coli Dilution and Bacteria MDA Test[edit]

Objective[edit]

  • Perform the E. coli dilution using Nanodrop.
  • Optimize the Bac MDA condition using different denaturing time (3min or 6 min).




Reagents[edit]

  • 2-mL microcentrifuge tubes
  • 1X PBS (sterile)

Procedures[edit]

E. coli enrichment and dilution[edit]

  • Warm up the LB broth to RT (sterile, storedin 4 degree fridge). Prepare two 250 mL flask and filled with 50 mL LB broth. Inoculate 50 uL of E. coli in 50 mL LB both (1:1000 ratio).


  • Mix the broth and cap the flask-top with foil. Incube the flask in 37-degree incubator overnight.


  • The next day. Harvest the enriched E. coli by transferring 2 mL culture into 2-mL microcentrifuge tubes. Prepare 8 tubes and centrifuge at RT, 4000 rpm for 5 min to pellet the bacteria.


  • Remove the supernant. Perform PBS washing (using 1 mL PBS) and concentrating the pellet during each PBS washing
  • Measure the optical density using Nanodrop (chose 'cell-cultur' function, measure 1.5 uL E. coli suspension using OD600 (default) and OD590 (custom) nM wavelength.


  • Make sure to blank Nanodrop using PBS (the media used in BAC dilution). Try to adjust the O.D600 = 1.0, which should give a bacteria number around 10^9 CFU/mL = 10^6 CFU/uL


  • Dilute the BAC in series dilution as following:
      Dilution Factor     Start      1/100    1/1000    1/10000   1/100000
      --------------------------------------------------------------------
      BAC mixture         10 uL      10 uL     10 uL      10 uL      10 uL
      PBS                           990 uL     90 uL      90 uL      90 uL
      --------------------------------------------------------------------
      Concentration       10^6/uL   10^4/uL  10^3/uL     10^2/uL     10/uL
      ** Mix by repeat pipetting using p200 pipettor in each step

Bacteria MAD reaction[edit]

Exp. Design (total = 16 rxns)[edit]

Denature:                   3 min ALS Denature               6 min ALS Denature
                      ----------------------------        -------------------------
Bac. amount (cell)    1000    100     10     H2O          1000   100     10    H2O     
                      ----------------------------        -------------------------
Duplicate: Jeff   J-3  #A      #B     #C      #D      J-6  #E     #F     #G     #H     
            Sam   S-3  #A      #B     #C      #D      S-6  #E     #F     #G     #H


Preparation[edit]

  • Thaw all of the reagents in Phi29 enzyme kit - except the phi29 enzyme


  • Thaw N6-primer (200 uM)


  • Prepare fresh 5M KOH (MW=56.11), and make ALS buffer: 82 uL H2O + 10 uL 1 M DTT + 8 uL 5M KOH in a 1.5-mL tube


  • Test the pH of ALS and NS (used the one previously made) mixture(10 uL ALS + 10 uL NS). The acceptable pH is around 7~8
    • The complete mixing by pipetting is critical in this step


  • UV-treatment on ALS and NS for 10 min


  • 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: 85 C, 3 min (to kill all of the enzymes left)
         Line 5: 4 C, forever
         Line 6: End

Procedures[edit]

  • Preparing the reaction master mix: - for 16+2 rxns. Leave on ice for later use.


                                            1 rxn              18 rxn
         --------------------------------------------------------------
            H2O                             6.2 uL             111.6 uL
            10X Repliphi phi-29 buffer      2.0 uL              36.0 uL
            200 uM N6 primer                5.0 uL              90.0 uL
            25 nM dNTP                      0.8 uL              14.4 uL
            2X SYBR Green I                 1.0 uL              18.0 uL
            Repliphi Phi-29 (100 U/uL)      1.0 uL              18.0 uL
         --------------------------------------------------------------
                                           16.0 uL             288.0 uL  (288/18=16)


  • Prepare two stips of PCR tubes and transfer 1 uL of diluted Bacteria or H2O into each of PCR tubes.


  • Add 1.5 uL ALS into the tube and mix by pipetting. Incubate at RT for 3 min or 6 min (This step is done in RT)


  • Add 1.5 uL NS into the tube and mix by pipetting. Transfer the reaction on cool rack.


  • Add 16 uL phi29 master mix into each of tubes. Mix by pipetting and quick spining down. Cap and label the tube.


  • Place the reaction in Bio-Rad realtime PCR machine and start the reaction program (save as 'sam022009').


Results[edit]

FIG. Quantification of Realtime amplication

  File:Sam022009-bac-MDA.bmp File:Arrangement-sam.bmp
  B1->B4: Bac number from high to low conc. (1000/uL, 100/uL, 10/uL, H2O. ALS treatment 3 min)
  B9->B12: Bac number from high to low conc. (1000/uL, 100/uL, 10/uL, H2O. ALS treatment 6 min)

Discussion[edit]

  1. The bacteria DNA could be extracted by ALS treatment under the MDA reaction we set up.
  2. Similar results were found in ALS 3 min and 6 min treatments.
  3. The minimun amount of becteria is aound 10 cell/rxn in the current practice. The 1 cell/rxn should be done more carefully to prevent contamination.

Suggestion[edit]

  1. The other way to disrupt Bacteria before ALS treatment could be 1) heat and 2) Lysozime (epicenter) using 1 unit - treatment.
  2. Confirm bacteria number of bacteria dilution by plating.
  3. Confirm the amplified Bac genome using 16S primers we designed previously.