NOTEBOOK
Agarose Gel Electrophoresis
1. Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel).
2. Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave).
3. Let the solution cool down to about 40-50℃ and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb.
4. Let the gel cool until it becomes solid.
5. Pull out the comb carefully.
6. Place the gel in the electrophoresis chamber.
7. Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel.
8. Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel.
9. Run the gel at 135V for about twenty minutes.
Basic ELISA Protocol
There are many different types of ELISAs, which can detect the presence of protein in serum or
supernatent. One of the most common types of ELISA is the so-called "sandwich ELISA." It is termed this because the antibody that you are detecting gets sandwiched between an antigen and a chromogenically-conjugated antibody. Below you will find a basic protocol for this assay.
1、To coat the plate with the appropriate antigen, fill the microwells of a Immuno Plate with 100uL of the diluted antigen.
2、Incubate at 4℃overnight
3、Wash the unbound antigen off the plate by flicking the contents of the plate into the sink, fill the wells with PBS-Triton, flick again, repeat 2X with PBS-Triton.
4、Block non-specific binding by adding 200uL of 1%BSA/PBS
5、Incubate for 30-60minutes at Room Temperature (RT)
6、Wash plate as above
7、Add 100uL of (diluted) samples to appropriate wells. Be sure to include positive and negative controls, and, if necessary, a standard curve.
8、Incubate for 1hour at RT
9、Repeat washing step
10、Prepare appropriate dilution of the second step antibody conjugated either with Alkaline Phosphatase or Horseradish Peroxidase. (antibodies should be titrated for optimal dilution)
11、Add 100uL of second step antibody to wells and incubate for 1hr
12、Repeat washing step
13、Prepare substrate solution*
14、Add 100uL of substrate to well and incubate at RT for 60min
15、add stopping solution if appropriate
16、Read plates on an ELISA plate reader
*Common Substrates and the appropriate plate reader setting
ABTS: 405-410 nm
TMB: non-stopped 620-650 nm, stopped 450 nm
OPD: non-stopped 450 nm, stopped 490 nm
pNPP: 405-410 nm
BluePhosTM: 595-650 nm
Bind with NTA-Ni beads
1. To harvest overnight induced E. coli (OD: 4.0)200ul. 4000rpm centrifuge 1min and remove the upper cleaning fluid.
2. Use PBS to clean the bacteria in the EP tube twice, and use 250ul PBS to resuspent the bacteria.
3. Cells were fixed by mixing 250 ul cells with a 750 u1 4 % (w/v)solution of paraformaldehyde in PBS. This mixture was incubated on ice for 20 min.
4. To remove the fixative, cells were washed twice in PBS. and use 250ul PBS to resuspent the bacteria.
5. Adding primary antibody(monoclonal) ,1:2000 dilution, at 4℃incubation for one hours.
6. Cells were washed twice in PBS. and use 250ul PBS to resuspent the bacteria.
7. Adding second antibody(FITC-conjugated),1: 5 000 dilution, at 4℃ incubation for one hours.
8. Use PBS to clean the bacteria in the EP tube twice, and use 250ul PBS to resuspent the bacteria.
9. Adding 100ul NTA-Ni (50% slurry) to EP tube, NTA-Ni were washed twice in PBS. and use 50ul PBS to resuspent the NTA-Ni.
10. The NTA-Ni and bacteria cells avoided the light for incubation two hours.
11. The NTA-Ni and bacteria cells were washed twice in PBS, and use 250ul PBS to resuspended.
12. Gravity naturally subsidence for half an hour, sent to microscopic examination for precipitate.
cation- exchange chromatography
Sample preparation
For optimal growth, induction and cell lysis conditions, please refer to established protocols.
The sample should be fully dissolved. To avoid column clogging, we recommend centrifugation and filtration through a 0.22 μm filter to remove cell debris or other particulate material.
Purification procedure for a packed column
1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water.
2. Equilibrate the column with 5-10 column volumes of binding buffer. or until the column effluent shows stable conductivity and pH values.
Bind the sample
1. Add 10 ml of sample to 1 ml of the 50% slurry. Binding capacity of SP Sepharose Fast Flow
is protein dependent and the average is 40 mg/ml. This means that 1 ml of the 50% slurry can bind about 20 mg of total protein.
2. Incubate sample and the SP Sepharose Fast Flow slurry at room temperature on a shaker at low speed for 1 h.
3. Load sample/SP Sepharose Fast Flow mix onto the 1ML columns and collect flowthrough.
Buffer wash and elution
1. Wash with 5 × 1 ml of binding buffer and collect both fractions.
2. Elute with 4×1ml of elution buffer and collect the eluted fractions in four separate tubes.
Regeneration
After each separation, elute any reversibly bound material either with a high ionic strength solution(1 M NaCl in buffer) or by increasing pH. Regenerate the medium by washing with at least 5 bed volumes of buffer, or until the column effluent shows stable conductivity and pH values.
Measure absorbance at 280 nm using a spectrophotometer and confirm purity of pooled fractions by SDS-PAGE. Use elution buffer as the blank.
Store
Store the beads in 20% EtOH.
|
Wash/Bind Buffer (25 ml) |
Elution Buffer (10 ml) |
Regeneration Buffer (10 ml) |
|
50 mM Tris-HCI pH 6.4
|
50 mM Tris-HCI pH 7.5
500 mM NaCl |
50 mM Tris-HCI pH 6.0
1 M NaCl |
reagent
Ni affinity chromatography
Sample preparation
For optimal growth, induction and cell lysis conditions, please refer to established protocols.
The sample should be fully dissolved. To avoid column clogging, we recommend centrifugation and filtration through a 0.22 μm filter to remove cell debris or other particulate material.
Purification procedure for a packed column
1. If the column contains 20% ethanol, wash it with 5 column volumes of distilled water.
2. Equilibrate the column with 5-10 column volumes of binding buffer. or until the column effluent shows stable conductivity and pH values.
3.To prevent binding of host cell proteins with exposed histidine, add the same concentration of imidazole to the sample as to the binding buffer.
Bind the sample
1. Add 5 ml of sample to 1 ml of the 50% slurry. Binding capacity of Ni Sepharose 6 Fast Flow
is protein dependent and the average is 20 mg/ml. This means that 1 ml of the 50% slurry can bind about 10 mg of target protein.
2. Incubate sample and the Ni Sepharose 6 Fast Flow slurry at room temperature on a shaker
at low speed for 1 h.
3. Load sample/Ni Sepharose 6 Fast Flow mix onto the 1ML columns and collect flowthrough.
Buffer wash and elution
1. Wash with 5 × 1 ml of binding buffer and collect both fractions.
2. Elute with 4×1ml of elution buffer and collect the eluted fractions in four separate tubes.
Regeneration
1.After each separation, Regenerate the medium by washing with at least 5 bed volumes of buffer, or until the column effluent shows stable conductivity and pH values.
2.Measure absorbance at 280 nm using a spectrophotometer and confirm purity of pooled fractions by SDS-PAGE. Use elution buffer as the blank.
Store
Store the beads in 20% EtOH.
|
6XHis Wash Buffer (25 ml) |
6X His Elution Buffer (10 ml) |
6X His Regeneration Buffer (10 ml) |
|
50 mM Phosphate Buffer pH 7.0
|
50 mM Phosphate Buffer pH 7.0 |
50 mM Phosphate Buffer pH 7.0
|
reagent
DNA purification/Axygen gel extraction
1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination.
2. Absorb the liquids left on the surface of the gel slices using paper towels. Weigh gel slice (tare with empty tube).
3. Add 3 volumes of DE-A buffer per mg of gel (so a 100mg gel gets 300ul of buffer).
4. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved (keep heating for 6-8 minutes). If low-melt agarose gel is used, you may heat it at 40℃. Intermittently vortexing every 2-3 minutes will do a lot of help to accelerate the solubilization.
Note: Buffer DE-A is red liquid, so you can observe the color to make sure the gel is fully dissolved.
5. Add 0.5× Buffer DE-A volume of Buffer DE-B and mix. If the DNA fragment is less than 400bp, supplement further with a 1×sample volume of isopropanol.
Note: After the addition of DE-B, the solution should be in the uniform color of yellow.
6. Place a Miniprep column into a 2ml microfuge tube (provided). Transfer the solubilized agarose from the step above into the column. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
7. Return the Miniprep column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.
8. Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.
9 Place the Miniprep column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
10. Place the Miniprep column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.
11. Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 50ul of ddH2O to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.
Note: Pre-warm the ddH2O at 65℃ will generally improve elution efficiency.
Dot-blot
1. Spot 1-2 microliter of antigen on to a piece of membrane(Nitrocellulose nc membrane), let air dry for 30 min or longer;
2. Incubate with Blocking buffer for 8-12 hr;
3. Wash with wash buffer, 3x10min;
4. Incubate with primary antibody,1 hr;
5. Wash with wash buffer, 3x10min;
6. Incubate with enzyme-labeled secondary antibody, 30 min;
7. Wash with wash buffer, 3x10 min;
8. Add enzyme substrate, wait 5 min;
9. Detect by eye or with colorimetric or chemiluminescent imaging system.
|
Blocking Buffer |
Wash Buffer(1L) |
Antibody Dilution Buffer |
|
5% Skim milk powder 0.5% BSA |
8g NaCl 0.2g KCl 3g Tris-base 1ml Tween-20 PH=7.4 |
2.5% Skim milk powder 0.5% BSA |
E. Coli calcium chloride competent cells
1.Inoculate a single colony into 5ml LB in a 50ml Falcon tube. Grow overnight at 37°C.
2.Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.
3.Shake at 37°C for 1.5-3hrs. OR Inoculate a single colony into 25ml LB in a 250 ml bottle in the morning. and then shake at 37°C for 4-6 hrs.
4.Put the cells on ice for 10 mins (keep cold form now on).
5.Collect the cells by centrifugation for 3 mins at 6000 rpm.
6.Decant the supernatant and gently resuspend on 10 ml cold 0.1M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
7.Incubate on ice for 20 mins
8.Centrifuge for 3 minutes at 6000 rpm.
9.Discard supernatant and gently resuspend on 5ml cold 0.1 M CaCl2/15% Glycerol
10.Dispense in microtubes (300μl/tube). Freeze in - 80°C.
CaCI2/15% Glycerol-solutions for competent cells Material
0.1 M CaCl2
15 % glycerol solution
Fixation of bacterial cells and fluorescence labeling
1.Cells from overnight cultures (induced if required) were harvested and washed in PBS (0.145 M NaCl; 0.15 M sodium phosphate).
2.Cells were fixed by mixing 250 ul cells with a 750 u1 4 % (w/v)solution of paraformaldehyde in PBS. This mixture was incubated on ice for 20 min.
3.To remove the fixative, cells were washed twice in PBS.
4.Samples of 20 ul were placed on a poly-L-lysine-coated slide and air-dried.
5. After washing in PBS, 16 u1 of a 1: 5 (monoclonal) or 1: 25 (polyclonal) dilution of the primary antiserum was placed on top of each sample and left in a moist incubation chamber for 1h.
6.The slides were washed three times in PBS and 16 ul FITC-conjugated antiserum was added.
7.After 2 h in the dark, the slides were washed three times in PBS, and a drop of Citifluor was placed on top of each sample before microscopy.
General Heat-Shock Transformation
1. Add 10ul DNA to 50ul cells on ice (set positive control by using Pcotc, cotc, PtasA, GolB,PbrR DNA fragment and ddH2O, set negative control by using chemically competent E.coli cells without plasmids).
2. Incubate on ice for 30 minutes.
3. Heat shock at 42℃ for exactly 90 seconds.
4. Place samples back on ice for 1-2 minutes.
5. Operating in the clean bench, add 900ul of LB broth per tube.
6. Incubate at 37℃ for 60 minutes, shaking.
7. Activate it on the plate for 60 minutes. The total number of plates is 7.
8. Centrifuge at 3000rpm for 1 minute.
9. Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.
10. Use the inoculating loop to load bacteria liquid then streak on the LB plate.
11. Place plates upside down and incubate at 37℃ overnight.
LB medium
1.Dissolve 10 g peptone, 5 g yeast extract, and 5 g NaCl in 950 mL deionized water.
2.Adjust the pH of the medium to 7.0 using 1M NaOH and bring volume up to 1 liter.
3.Autoclave on liquid cycle for 20 min at 15 psi. Allow solution to cool to 55°C, and add antibiotic if needed (34µg/ ml of Amp or Kan).
4.Store at room temperature or +4°C (2).
Material
10 g peptone
5 g yeast extract
5 g NaCl
1 M NaOH
Antibiotic if needed
dH2O
LB agar-plates
1.Prepare LB medium as above, but add 10 g/L agar before autoclaving.
2.After autoclaving, cool to approx. 55°C, add antibiotic (if needed, the concentration of antibiotic to LB should be 1:1000), and pour into petridishes.
3.Let it cool, then invert and store at +4°C in the dark (2).
Material
LB medium
10 g/LAgar
PCR method/Taq PCR
1. Thaw Taq, dNTP, primers, template DNA on ice.
2. To a new PCR tube, add:
|
template DNA |
0.5ul |
|
dNTP |
2ul |
|
10×buffer |
2.5ul |
|
Mg2+ |
2ul |
|
F primer |
1ul |
|
P primer |
1ul |
|
rTaq E |
0.5ul |
|
ddH2O |
15.5ul |
|
total |
25ul |
3. Mix solution well.
4. Place tube in PCR thermocycler. Set thermocycler program:
Inititial denaturation: 3min at 95℃;
Loop (29 cycles),
Denaturation: 30s at 95℃,
Annealing: 30s at 60℃,
Elongation: 1min at 72℃;
Final elongation: 10min at 72℃;
Store: 12℃.(not for too long).
5. We use 5ul of the PCR product for electrophoresis and 45ul for purification (details see DNA purification/AxyPrep PCR DNA purification PCR).
Plasmid extraction
1. Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely.
2. Add 250ul of Buffer S1 to the pellet to resuspend bacteria cells.
3. Add 250ul of Buffer S2, mix gently by inverting the tube 4-6 times until the solution becomes clear. The time should be no longer than 5 minutes.
4. Add 350ul of Buffer S3, mix gently by inverting the tube 6-8 times.
5. Centrifuge at 12,000rpm for 10 minutes.
6. Place spin column into a 2ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.
7. Return the column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
8. Return the column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
9. Place the column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
10. Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.
11. Transfer the column into a clean 1.5ml microfuge tube (provided). Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.
Preparation of single cell suspension of spleen tissue
Cut up method：
1.Add a small amount of PBS solution and 20% fetal bovine serum after putting the tissue into a plate.
2.The tissue was cut to homogenate by eye scissors, and 5mL of PBS fluid and 20% fetal bovine serum were added.
3.Use straws to absorb tissue homogenized and filter into test tube with 100 mesh stainless steel strainer (other purchase).
4.The centrifuge precipitates 1, 500 rpm x 3 min,and then cleaned 3 times with the cell cleaning fluid.
5. Each time, the cell debris is removed by 500 rpm short and low speed centrifuge, and the cell block is filtered by 200 mesh stainless steel strainer (other purchase).
6.The cell count and the adjustment of cell concentration were (2 ~ 5) x 107 / mL.
7.Place at room temperature to measure the activity of cells.The cell concentration was 2 x 108~1 x 109 /mL of single-cell suspension with 20% fetal bovine serum or sample diluent before separation.
protein’s inducing
1. For the engineered-competent E.coli cells stored at -80℃, culture them on LB-agar medium using streak plate method.
2. After culturing on LB-agar at 37℃ for 12 hours, pick a single colony, shake in 3mL LB liquid buffer, 37℃, 160rpm, for 8 hours over night.
3. Extract 1mL from 300mL sanitized LB liquid buffer, measure its OD600 as blank. Add the 3mL product of step ii into ~300mL LB liquid buffer.
4. Shake for about 2 hours, 37℃, 160rpm, until OD600 reaches ~0.6 (OD600 should be measured after 1.5 hours and could be predicted using the formula OD600t+20min=2*OD600t).
5. Add 120uL 1M IPTG. to a final concentration:1-4 mM. After culturing at 37℃ for 12 hours.
SDS-PAGE Electrophoresis
According to the size of the target protein, select the appropriate concentration of PAGE separation gel. Table 1 list gel concentration formula.
I Preparation of separation gel
1. Different volumes of pure water, 30% Acrylamide, 1.5M Tris-HCI Buffer (PH=8.8)and 10%SDS was added to the centrifuge tube.
2. Add 10% APS and TEMED, and mix immediately for 5-10 seconds to allow the solution to mix well.
3. In the gel mold, quickly poured into the appropriate amount of separation glue solution (for 1 mm mini-gel, separation gel solution plus about 4 ml)
And then gently cover a layer of 1-3 cm the water layer, so that the gel surface remained flat.
4. A clear interface between the gel and the water indicates that the gel has polymerized.
II Preparation of concentrated gel
Remove the water layer covering the interlayer gel and drain the remaining water with filter paper.
1. Different volumes of pure water, 30% Acrylamide, 1.5M Tris-HCI Buffer (PH=8.8)and 10%SDS was added to the centrifuge tube.
2. Add 10% APS and TEMED and mix immediately for 5-10 seconds to allow the solution to mix well.
3. Insert the comb into the gel to avoid air bubbles.
4. After gel polymerization, carefully pull out the comb to avoid damaging the fill holes.
5. Perform electrophoresis.
IV Electrophoresis
The electrophoresis tank into the 4℃ or ice bath, the outer tank by adding SDS buffer, the inner tank into the SDS buffer, after adding the sample, 250V electrophoresis 5 min, 110V electrophoresis to bromophenol blue to reach the bottom of the gel. After the stop electrophoresis, carried out Coomas bright blue staining or electric transfer.
