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]
- The bacteria DNA could be extracted by ALS treatment under the MDA reaction we set up.
- Similar results were found in ALS 3 min and 6 min treatments.
- 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]
- The other way to disrupt Bacteria before ALS treatment could be 1) heat and 2) Lysozime (epicenter) using 1 unit - treatment.
- Confirm bacteria number of bacteria dilution by plating.
- Confirm the amplified Bac genome using 16S primers we designed previously.