Sam:LabNotes/Microbione/2009-2-20

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Revision as of 00:37, 23 February 2009 by >Sam Chiang (New page: ='''E. coli Dilution and Bacteria MDA Test'''= ==Objective== *Perform the E. coli dilution using Nanodrop. *Optimize the Bac MDA condition using differnt denaturing time (3min or 6 min)...)
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E. coli Dilution and Bacteria MDA Test

Objective

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




Reagents

  • 2 mL tubes
  • 1X PBS (sterile)


Procedures

E. coli enrichment and dilution

  • Warm up the LB borth to RT (sterile, storedin 4 degree fridge). Prepare two 250 mL flask and filled with 50 mL LB broth. Innoculate 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 transfering 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 twice (using 1 mL PBS)


  • Remove the supernatant, repeat the PBS washing one more time.


  • After two PBS-washing, resuspend pellet with 0.5 mL PBS, mixed the suspension from few tubes into one to concentrate the E. coli suspension.


  • Measure the optical density using Nanodrop (chose 'cell-cultur' function, measure 1.5 uL E. coli suspension using OD600 (default) and OD590 (custom) nM wave-lenth.


  • 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

Exp. Design (total = 16 rxns)

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

  • 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 acceptible 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

  • 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

FIG. Quantification of Realtime amplication

  1. A->E: Template from high to low conc. (30 pg/uL, 3 pg/uL, 300 fg/uL, 30 fg/uL and 3 fg/uL, respectively)
  2. F->H: Blank control.

Discussion

  1. The weak flourescent signle suggested that the amplification was not successul.
  2. The The difference of samples with different template conc. wasn't distinguishable in different curves.
  3. The non-smooth curve may be due to the incomplete dissociation of dsDNA?

Suggestion

  1. Check the SYBR 2X reagent. The realtime exp on 02-04-09' showed unwanted curves too although the amplification was successful (confirmed by agarose gel).
  2. Check the primer concentration (100 uM for N6-primer should be used?)
  3. Provide enough time for ALS treatment, and shorten the procedure time on after we nutralize the ALS with NS.