Preparation of Competent Cells

1. 1. Take 100mL aliquot of frozen cells (use DH5-α cells in this case) from the -80°C and inoculated.
2. 2. Grow the cells in the shaker at 37°C until they reach an O.D.600nm=0,6.
3. 3. Transfer them into sterile Falcon (50mL).
4. 4. Centrifuge it at 4500 rpm for 10 minutes at 4°C.
5. 5. Resuspend the bacteria pellet on ice in cold CaCl₂(0,1M) .
6. 6. Keep this suspension on ice for overnight.
7. 7. Centrifuge it at 4500 rpm for 10 minutes at 4°C.
8. 8. Resuspend the pellet in 8mL of RF2 (MOPS 1mM, RbCl 10mM, CaCl2 75mM, glycerol 15%w/v).
9. 9. Dispense in aliquots and freeze cells at -80°C.

Transformation - Heat Shock

1. 1. Take competent E.coli cells from –80°C freezer and place on ice. Allow cells to thaw.
2. 2. Mix cells by flicking the tube gently, then remove 100μl per transformation into a sterile pre-chilled (on ice) 1,5 tube.
3. 3. Add 7μl of DNA per 100μl cells. Quickly flick the tube several times to ensure the even distribution of DNA.
4. 4. Immediately place tubes on ice for 30 minutes.
5. 5. Heat shock the cells for 90 seconds in a water bath at exactly 42 °C. Do no shake.
6. 6. Immediately place tubes on ice for 2 minutes.
7. 7. Add 1mL of room temperature LB media (with no antibiotic added) and incubate for 1 hour in shaker at 37°C.Can incubate tubes for 30 minutes with appropriate antibiotic added – usually Ampicillin or Kanamycin.
8. 8. Spread about 100μL of the resulting culture on LB plates - Grow overnight (O/N). The cells may be pelleted by centrifugation at 500 x g for 5 minutes, then the cells can be resuspended and plated.
9. 9. Pick colonies about 12-16 hours later.

Clean colony PCR

1. 1. Pick the bacterial colonies and release them in 50μL distillate/autoclaved water.
2. 2. Boil the sample at 95°C for 5 minutes.
3. 3. Prepare 28μL of mix-PCR solution for each sample (6μL Buffer Taq 5x, 1,8μL MgCl2 25mM, 0,6μL dNTPs 5mM, 0,15μL per primers, 0,15μL Taq polymerase, 19,15μL H2O), blend it and then spin it.
4. 4. Take 2mL of the sample and release into mix-PCR solution and blend it.
5. 5. Impost the PCR machine for 30μL volume and for 30 cycles, 5 minutes at 93°C, 30 seconds at 95°C, 30 seconds at 53°C, 1 minute at 72°C, indefinitely at 4°C.
6. 6. Insert the samples and start the PCR machine.
7. 7. At the end of the PCR the samples are ready for electrophoresis.

E.L.I.S.A.

1. 1. Coat 96-well ELISA plate with 100μL per well of antibody I anti6HIS used 1μg/mL for selection. Coating is in 100mM sodium hydrogen carbonate, pH 9.6. Leave O/N at 25°C.
2. 2. Rinse wells 5x with BSA-PBS 0,1%.
3. 3. Add the bacteria transformed that express the 6HIS tag in different concentration: 10⁶, 10⁵, 10⁴ in different wells. Then in different wells too add bacteria non-transformed in different concentration: 10⁶, 10⁵, 10⁴.
4. 4. Rinse wells 5x with LB media.
5. 5. Add 200μL of LB media and possibly antibiotics. Incubate O/N at 37°C.
6. 6. Plate and incubate at 37°C until formation of bacterial colonies.

Western blot

1. 1. Inoculate bacteria in 20mL of LB media with antibiotics if required O/N.
2. 2. Transfer 2mL of the inoculum in flask and add 18mL of LB media with antibiotics if required.
3. 3. Grow the bacteria until the inoculum reach at O.D. 600nm the value 0,4-0,6.
4. 4. 2mL must be recovered to form the sample “non-induced”.
5. 5. Induce the remaining 17mL of inoculum with 17μL of IPTG.
6. 6. Wait for 4 hours (or for the time deemed appropriate).
7. 7. Take 2ml the induced and centrifuge it for 10 minutes at 5000 rcf.
8. 8. Discard the supernatant and add to the pellet 200μL of Loading Buffer SDS.
9. 9. Sonicate very strong.
10. 10. Heat shock at 95°C for 5 minutes.

SDS-PAGE

1. 1. Assemblate the Western blot scaffold.
2. 2. Seal the bottom of the Western blot scaffold using agarose-water solution.
3. 3. Add 10mL the running gel.
4. 4. Add immediately, before the gel get solid, 1mL of isopropanol to level off the gel surface.
5. 5. When the running gel is solid, add stacking gel until edge.
6. 6. Insert the comb and wait the solidification of the gel.
7. 7. Fill the Western blot scaffold with running buffer.
8. 8. Remove gently the comb and wash the wells with Loading buffer SDS.
9. 9. Loading the samples into wells.
10. 10. Run Western blot at 200V, 30mA for 2 hours.

1. 1. At the end of the SDS-PAGE, disassemble the Western blot scaffold and recover the gel.
2. 2. Assemblate the sandwich with in the middle the PVDF membrane surrounded two pieces of filter paper.
3. 3. Put the sandwich into transfer box, add transfer buffer.
4. 4. Leave transfer proteins at 200V, 50mA O/N.

Blocking
1. 1. Dip the PVDF membrane into PBS-milk 5%, 1 hours in shaker, to prevent non-specific binding of the antibodies, which leads to high backgrounds.
2. 2. Discard the PBS-milk 5% solution.
3. 3. Add Antibody I with PBS-milk 5% solution and incubate for 1 hours in shaker.
4. 4. Discard antibody with PBS-milk 5% solution.
5. 5. Wash three times with PBS-tween 0,1% solution shake manually.
6. 6. Wash three times with PBS-tween 0,1% solution in shaker for 3 minutes each one.
7. 7. Add antibody II with PBS-milk 5% solution and incubate for 1 hours in shaker.
8. 8. Repeat step 5 and step 6.

ECL (enhanced chemiluminescence)

1. 1. Dip the membrane in PBS.
2. 2. Put 1mL first ECL solution + 1mL second ECL solution on film.
3. 3. Wet the membrane in solution with the solution on film for some seconds.
4. 4. Fix the wet membrane in obscure box.
5. 5. Go to obscure room and lean the photograph plate on the membrane for the time deemed appropriate for to have the right exposure.
6. 6. Dip the photograph plate into developer solution for 1 minute.
7. 7. Wash the photograph plate with water.
8. 8. Dip the photograph plate into fixer solution for 2 minutes.
9. 9. Dry the photograph plate in stove at 65°C.

Stripping

1. 1. Wash the photograph plate with PBS-Tween 0,1% 3 times shake manually.
2. 2. Wash the photograph plate with distillate water.
3. 3. Add Stripping solution + distillate water in a ratio of 1 to 10 and incubate 30 minutes in shaker at 25°C.
4. 4. Repeat the point 1.
5. 5. Blocking with PBS-milk 5% for 30 minutes in shaker at 25°C.
6. 6. Repeat the point 1.
7. 7. Start the protocol for ECL (enhanced chemiluminescence).
8. 8. Should be a photograph plate without any signal.

Precipitation of Surnatant

1. 1. Recover 1mL of the sample supernatant.
2. 2. Add 250mL of TCA 50% (trichloroacetic acid).
3. 3. Incubate in ice for 60 minutes.
4. 4. Centrifuge it for 10 minutes at 3750 rfc at 4°C.
5. 5. Discard the supernatant.
6. 6. Add 1mL of could acetone to eliminate the TCA.
7. 7. Speed vaac for 10 minutes (sniff the sample to control absence of acetone).
8. 8. Resuspend the sample in sample buffer SDS 1X (the color change to yellow).
9. 9. Add 1mL of Tris (2-Amino-2-hydroxymethyl-propane-1,3-diol) to basific the solution (the color should change to blue, if it no happen so add other Tris).

Periplasm Protein Preparation

1. 1. Inoculate bacteria in 20mL of LB media and possibly antibiotics O/N.
2. 2. Transfer 2mL of the inoculum in flask and add 18mL of LB media and possibly antibiotics.
3. 3. Grow the bacteria until the inoculum reach at O.D. ₆₀₀=0,4-0,6.
4. 4. 2mL must be recovered to form the sample “non-induced”.
5. 5. Induce the remaining 17mL of inoculum with 17μL of IPTG mM.
6. 6. Wait for 4 hours (or for the time deemed appropriate).
7. 7. Take 2mL of the induced and centrifuge it for 10 minutes at 5000 rpm.
8. 8. Discard the supernatant and resuspend pellet in 1/40 volume of PPB buffer (200mg/mL sucrose, 1mM EDTA, 30mM Tris-HCl pH 8.0). Keep in ice for 20 minutes.
9. 9. Spin down cells in centrifuge at 5000 rpm for 15 minutes and collect supernatant into smaller high speed centrifuge tubes.
10. 10. Resuspend pellet in 1/40 volume of 5mM MgSO₄ buffer. Incubate in ice for 20 minutes.
11. 11. Transfer samples to small high speed centrifuge tubes.
12. 12. Spin both peri-prep supernatant and Osmotic shock preparation at 15000 rpm for 15 minutes.
13. 13. Collect the supernatants, the sample is ready for E.L.I.S.A.