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From 2014.igem.org

Contents 1 M13 1.1 Phage Infection 1.2 Plaque Amplification and Concentration 1.3 Western Blot 1.3.1 Sample Preparation 1.3.2 Protein Electrophoresis 1.3.3 Membrane Electrotransfer (Wet Transfer) 1.3.4 Immunoblotting

M13

Phage Infection

1.Pipette 200ul host bacteria culture(ER2738, with OD600=0.5) in the centrifuge tube. Bacteria should be in the pre-log phase. Usually, select a single colony from plates and then culture at 37℃ for 5-7 hours would be just ok.

2.Add 10-20ul phage solution into the host. Vortex and invert the tube and incubate in the room temperature for 5-10 min.

3.Pipette 200ul infected host cells into the upper agar. Attention, use the blue pipette tips and the top agar should be about 40℃.

4.Vortex and invert the top agar and pour it on the plate immediately.

Plaque Amplification and Concentration

1. Dilute an overnight culture of ER2738 1:100 in LB. Dispense 20 ml of diluted culture into culture tubes, one for each clone to be characterized. 10-20 clones from the third round are usually sufficient to detect a consensus binding sequence.

2. Use a pipette tip to stab a blue plaque from a tittering plate(important: plates should be <1-3 days old, stored at 4℃ and have <100 plaques) and transfer to a tube containing the diluted culture. Pick well-separated plaques. This will ensure that each plaque contains a single DNA sequence.

3. Incubate the tubes at 37℃ with shaking for 6 hours.

4. Transfer the cultures to microcentrifuge tubes, and microfuge at 5,000 g for 10 minutes. Transfer the supernatant to a fresh tube and microfuge at 9,000 g for 10 minutes again.

5. Pipette the upper 80% of the supernatant to a fresh tube and add 1/6 volume of 20% PEG/2.5 M NaCl. Allow the phage to precipitate at 4°C for at least 2 hours or overnight.

6. Spin the PEG precipitation at 9,000 g for 5 minutes at 4°C. Decant and discard the supernatant, re-spin briefly, and remove residual supernatant with a pipette.

7. Suspend the pellet in 1 ml of TBS. Transfer the suspension to a microcentrifuge tube and spin at 20,000 rpm for 5 minutes at 4°C to pellet residual cells.

8. Transfer 60% the supernatant to a fresh microcentrifuge tube and re-precipitate with 1/6 volume of 20% PEG/2.5 M NaCl. Incubate 15–60 minutes on ice. Microcentrifuge at 20,000 g for 10 minutes at 4°C. Discard the supernatant, re-spin briefly, and remove residual supernatant with a micropipet.

9. Suspend the pellet in 50 μl of TBS. Titer as described in Phage Infection. The titer should be approximately 10^14 pfu/ml. The phage stock can be stored for several weeks at 4°C. For long-term storage, add an equal volume of sterile glycerol and store at –20°C.

Alternative steps could be chosen after step 3. Centrifuge bacteria culture at 12000g for 15~20 min. Take the supernatant and filtrate it with 450nm filter membrane. Filtrate again with 220nm membrane and collect the supernatant.

Western Blot

Sample Preparation

<p>1. Enlarging culture in 37℃ overnight.

2. Dilute a 2 hours culture 1:100 in LB.

3. Add Arabinose to induce for 5 hours.

4. Collect all the bacteria and centrifuge 5000g for 10 min.

5. Add PBS to resuspend, then adjust and assure that the OD are the same.

6. Ultrasonication for 3 min, then centrifuge 14000g for 10 min.

7. Separate the precipitation and supernatant, use PBS to resuspend the precipitation.

8. Add loading buffer and heat at 100°C for 10 min.

Protein Electrophoresis

1. Prepare your SDS-PAGE gel by inserting it into the electrophoresis apparatus and filling with running buffer. Rinse the wells of the gel with running buffer and verify there are no bubbles.

2. Load the appropriate amount of sample into each well. If you are unsure of the amount to load, 10-30 µg of total protein is a suggested starting point.

3. Load prestained molecular weight ladders in the first and last lane in order to monitor the separation during electrophoresis, and subsequently verify protein sizes during analysis.

4. Run the gel until the loading buffer reaches the bottom. This is typically 45-60 minutes at 200 V (consult manufacturer’s guidelines).

Membrane Electrotransfer (Wet Transfer)

1. Remove gel from the cassette and float in transfer buffer.

2. Choose either PVDF or nitrocellulose membrane for transfer (personal preference). PVDF needs to first be activated in methanol for 2 minutes. Incubate membranes in cold transfer buffer for 10 minutes.

3. Set up gel/membrane sandwich by placing the transfer cassette in cold transfer buffer. Create a sandwich stack by placing the following components from the cathode (negative black side) to the anode (positive red side) so that the negatively charged proteins move from the gel into the membrane.

4. (- side) sponge, filter paper, gel, membrane, filter paper, sponge (+ side)

5. Use a clean roller with each layer to gently roll out any bubbles present.

6. Lock the cassette and place in the transfer apparatus containing cold transfer buffer. Perform the transfer according to the manufacturer’s instructions (normally 100 V for 30-120 minutes). In order to prevent heat buildup, it is beneficial to transfer with a cold pack in the apparatus, or in the cold room with a spinner bar.

Immunoblotting

1. Remove the membrane from the cassette and wash three times in ddH2O.

2. Incubate the membrane with blocking solution for 1 hour at room temperature. Common blocking buffers include 5% non-fat dry milk, or 3% BSA, in TBS-T (1x TBS, 0.1% Tween-20). Do not use milk when probing with phospho-specific antibodies.

3. Decant blocking solution and wash with TBS-T for 5 minutes.

4. Dilute the primary antibody in blocking buffer at the concentration recommended on the datasheet. Incubate membranes with primary antibody overnight at 4°C on with gentle shaking.

5. Decant primary antibody and wash membrane with large volumes of TBS-T and vigorous agitation five times for 5 minutes each.

6. Dilute the secondary antibody in blocking solution and incubate the membrane for one hour at room temperature at the concentration recommended on the datasheet. The secondary should be directed against the host species of your primary antibody. E.g. If using a primary that was raised in rabbit, the appropriate secondary would anti-rabbit. Most secondaries are conjugated to an enzyme such as HRP in order to catalyze the electrochemiluminescent (ECL) reaction.

7. Decant secondary and wash membrane with large volumes of TBS-T and vigorous agitation five times for 5 minutes each.

8. Membrane can be visualized by DAB reagents (1:1) according to the manufacturer’s instructions. Incubate the membrane for 3-5 minutes and use water to stop the reaction properly.