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Protocols (in alphabetical order)

Bacteria Lysis

1. Harvest the culture by centrifuge (13krpm*1.5min) followed by washing with 1ml PBS twice. Later steps should be done on the ice.
2. Re-suspend the pellet with PBS and protease inhibitor cocktail.
3. Sonication for (5.5seconds+1second pulse)*10 minutes and protein solutions are obtained.

BCA Quantification Analysis

1. Prepare a working solution of BCA reagent just prior to use by adding BCA Reagent A and BCA Reagent B with a ratio of 50:1. Mix the two solutions until a clear green solution forms. Prepare the BCA working reagent fresh daily. 100uL is required for each sample.
2. For each protein determination, add 2~5uL protein sample and 100uL BCA mixture into each well. Usually each sample will be loaded into 3 wells to reduce errors.
3. Put the 96-well plate in the warm room for about 30 minutes.
4. Read the wells in a suitable plate reader (e.g. Molecular Devices) at the wavelength of 562 nm.

Competent Cell Preparation for Electro Transformation

• Materials
  1. Media: LB (Both liquid media and media containing agar. Add certain antibiotic if it is necessary.)
  2. Buffers and Solutions: 10% Glycerol.
  3. Special Equipments : EP tubes (1.5mL), micropipette tips, centrifugation bottles (polypropylene tubes, 50mL), graduated flask (250mL*2, 5mL*1), plates and test tubes.
• Steps
  1. Sterilization
     1. Including all materials in part2.
     2. Caution (Remind): use some special marks to distinguish the sterilized materials from the unsterilized ones.
  2. Preparation after sterilization
     1. Chill the 10%Glycerin to 4 centigrade degree
     2. Decant LB containing agar into the plates.(If antibiotics are necessary, be sure that they are added when the media temperature is below 60 centigrade degree.)
  3. Streak the prepared strains onto the agar plates. Then incubate it at 37℃ for 10-16 hours.
  4. Overnight pre-culture
  5. Take 0.5mL overnight culture to 50mL LB bottle.
  6. 37℃ shaking 100~120 min to O.D. 600=0.45~0.6
  7. On ice for 30min
  8. 4000rpm 7min at 4℃
  9. Add origin volume 10% glycerol, suspend softly.
  10. Repeat the step 7,8
  11. Add origin volume 1/10 10% glycerol, suspend softly.
  12. 4000rpm7 min at 4℃
  13. Add origin volume 1/100 10% glycerol, suspend softly, store at -80℃.

Electro Transformation

1. Hold competent cells (from -80℃ refrigerator) on ice.
2. Gently mix ligation product (1-5 μL) with cells.
3. Transfer the cell/DNA mix into an electroporation cuvette.
   Note: the gene pulser should already be set properly
   • time constant = 4.5 - 5.0 ms
   • resistance = 200 W
   • capacitance = 25 mFD for 0.1 cm gap cuvettes, set the volts to 1.8 kV
4. Pulse the cells once; the voltage display blinks, and the gene pulser beeps
5. Quickly transfer 37 ℃ SOC to cuvette, mix by gently pipetting up and down, and transfer SOC/cells back to culture tube.
6. Bath in 37℃ for 30~60 min.
7. Separate cells on petri-dishes, and cultivate them in 37℃ for 12-16 hours.

Ligation

England Biolabs T4 DNA ligases are used here.

1. Choose reaction volume: 5-10 μL
2. Mix proper proportion (usually 3~5:1) of DNA fragment and vector.
3. Add 10 × ligase buffers.
4. Add 0.5 μL ligase per 10 μL final volume.
5. Bath in 16℃ water for 12 hour,
6. Begin transformation.

Membrane Biotinylation

1. Activate the membrane with methanol for 10-30seconds
2. Balance the membrane in PBS for 5 minutes
3. Place the membrane on a piece of filter paper.
4. Drop the protein-biotin complex onto the membrane.
5. Air-dry for 5 minutes.
6. Soak in methanol for 1minutes.
7. Place the membrane on a piece of filter paper.
8. Air dry for 15 minutes.

Pre-culture

1. One single colony is picked up from the agar plate and transferred to a tube.
2. Add 3~5mL LB broth to the tube and the specific resistance.
3. Culture overnight at 32℃ or 37℃.

Recombineering

1. Overnight cultures: 5mL medium (containing antibiotic where applicable) from single colonies grown at 32°C for 18 h.
2. Get 500uL from overnight cultures expanded into 50 ml of L medium in a 250 ml Erlenmeyer flask, and incubated at 32°C for 2 h (until OD600 of ca. 0.4–0.6).
3. Flasks were transferred to a shaking water bath at 42°C and incubated for 14–15 min, before cooling to 0°C as rapidly as possible in iced water.
4. After 15–20 min, cells were harvested by centrifugation at 0°C (4000 g, 9 min).
5. Cell pellets were carefully washed three times with sterilized ice-cold water (2 × 50 ml, then 1 × 1.5 ml) then re-suspended in 100–200 μl of ice-cold water.
6. Competent cells (50 μl) were transformed with 50–200 ng of (gel purified) linear dsDNA targeting cassette using a BioRad electroporator (1.8 kV, 25 mF, 200 W).
7. The LB medium (1 ml) was added to the transformed cell mixture, which was incubated at 32°C, for 2 h. Cells were collected by centrifugation, ca. 900 μl of supernatant media was discarded, and then the re-suspended cells were plated onto LB agar containing the appropriate antibiotic to select for resistant colonies. Reference to Watt et al [1].

SDS-PAGE and Western Blotting

1. Buffer preparation
   • Rususpension buffer: 1ml stock + 0.1ml protease inhibitor (10x)
   • Loading buffer: 1ml stock + 0.2 ml DTT + Phenol Blue
2. Sample treatment
   • centrifuge the culture into pellet (13k rpm 10min)
   • Resuspend the pellet with 5xVolume(pellet) Resuspension buffer
   • Add 5xVolume(pellet) loading buffer
   • Boil the resuspend for 10 minutes at 100’C
   • Centrifuge for 1 min at 13krpm
3. SDS-PAGE
   • 15% separation gel
   • 5% stacking gel
   • Load the sample and run under the constant voltage of 100V.
   • Visualize your proteins using Coomassie Brilliant Blue, Silver stain, or any of the other protein stains or blot the gel for western blotting.
4. Western blotting
   • Transfer from gel to membrane
   • Assemble "sandwich" Transblot.
   • Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer.
   • Transfer for 1 hr at 1 amp at 4°C on a stir plate. Bigger proteins might take longer to transfer. For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer. When finished, immerse membrane in Blocking buffer and block overnight.
5. Hybridization with antibodies
   • Incubate with primary antibody diluted in Blocking buffer for 60 min at room temp.
   • Wash 3 x 10 min with 0.05% Tween 20 in PBS.
   • Incubate with secondary antibody diluted in PBS for 45 min at room temp.
   • Wash 3 x 10 min with 0.05% Tween 20 in PBS.
   • Detect with SUPER SIGNAL WEST PICO Kit (1ml luminol solution + 1ml stable peroxide solution

• Result analysis
  • The size of the tagged protein can be determined by the marker (protein ladder)
  • The amount of the protein can be estimated by the brightness of the band, or accurately analyzed by the software.
• Trouble shooting
  • Unspecific binding : 1st antibody-membrane and irrelevant protein to 1st antibody
  • Film over-exposure or lack of exposure

Reference

1. Watt RM, Wang J, Leong M, Kung HF, Cheah KS, Liu D, Danchin A, Huang JD. Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes. Nucleic Acids Res. 2007, 35(6):e37.