Put blot into antibody:
- Remove blot from fridge.
- Prepare primary antibody:
- 10mls TBS
- 2% Non-fat powdered milk
- 0.05% Tween
- Add appropriate amount of antibody for dilution (1:5,000, 1:1,000, 1:500, etc.).
Prepare plastic bag:
- Cut plastic tube to size of gel.
- Seal bag with bag sealer set on 3.
- Put membrane blot in bag.
- Seal three sides of the plastic bag and cut excess (leave extra room on the top for when adding the antibody).
- Add antibody with pipette and aide.
- Completely wet membrane.
- Use a kimwipe to push out all the air bubbles.
- Seal bag closed once all bubbles are removed and cut bag excess.
- Put blot on rocker 1-2 hours at room temperature or overnight at 4 degrees Celsius.
- Take blot out of the bag and put in TBST rinse.
TBST (For 1 Liter):
- 900mls H2O
- 100mls 10X TBS
- 0.5mls Tween
- Save primary antibody. Store at 4 degrees Celsius.
- Put blot in tray with 100-200mls TBST and put on rocker for 3-5 minutes.
- Repeat rinse in TBST.
- Prepare secondary antibody (1:5,000):
- 25mls TBST
- 5ul secondary antibody
- Pour secondary antibody onto blot and put on rocker for at least 1 hour.
- Wash blot with 3 washes of TBST for 3-5 minutes each.
- Prepare Western Blotting Detection Solution:
- 3mls solution A
- 3mls solution B
- Lay glass plate down on bench and lay blot on glass plate.
- Put developer drop-wise over blots surface.
- Tilt blot to make sure the whole blot is covered.
- Let sit 1 minute.
- Lay out a piece of smooth seran wrap.
- Take blot off plate and let developer drip off.
- Lay blot protein side down onto seran wrap and fold seran wrap to seal blot in bag.
- Tape blot into cassette.
- Take Biomax light film, cassette, timer and scissors down to the dark room.
- Cut film into 2 pieces (fold upper right corner).
- Put film over blot and close cassette.
- Expose blot for 1 minute.
- Put film in developer.
- Expose blot for longer or shorter times depending on first exposure.
- Label film using blot (when it is still taped into the cassette)
II. Coomassie Blue Staining:
Materials and Reagents:
1. Coomassie Brilliant Blue R250 (EM Science) Equipment:
- Incubate the gel in staining solution with shaking for 30 min or longer (can leave it overnight).
- Remove the dye solution (it can be reused for many times) and rinse the gel with water 1-2 times to remove the dye.
- Add destaining solution to the gel and incubate for 30-60 min.
- Transfer the gel to water (can keep it in water for several days)
100 ml Staining solution
- Coomassie Brilliant Blue R250 0.25 g
- Glacial acetic acid 10 ml
- MetOH:H2O (1:1 v/v) 90 ml
Destaining solution is the same as staining solution, but not containing the Coomassie R250 dye Powder.
III. Silver Staining:
- Fix gel in 10% MeOH / 10% Acetic Acid for 30 min or overnight
- Wash 4X in H2O for at least 5 min
- Incubate in sodium thiosulfate (1-2 pellets [400mg] per 500 ml) for 90 sec (Save 20 ml for later)
- Wash 3X quickly with H2O
- Add Silver nitrate solution (0.9 g silver nitrate in 500 mL H2O) for 10 min. Gel will turn slightly yellow
- Wash the gel 3X quickly in H2O
- Add developer solution (10g potassium carbonate, 20 ml sodium thiosulfate, 250 μl 40% Formaldehyde in 500 ml)
- Stop the reaction by adding destain (10% MeOH and 5% Acetic Acid)
- Wash gel in water
- order two primers which are complements of one another.
- These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
- The "end primers" will not have any complements and will likely only have restriction sites.
- PCR amplify the necessary fragments separately. Use a proofreading polymerase enzyme. Use an annealing temp of 60°C.
- Clean up the product using a DNA column.
- "Overlap PCR" Use cleaned up fragments as template in a PCR reaction:
- About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments. Do not use Phusion polymerase. Try Pfu Turbo. Do not add any primers; the templates will prime each-other. 3.Run 15 PCR cycles without primers.Use an annealing temp of 60°C.
- Add end primers to the Overlap PCR reaction: Continue cycling for another 15-20 rounds. Use an annealing temp of 72°C
- Gel extract the correct size fragment.
V. Gel Extraction:
Method:QIAquick Gel Extraction Kit I(250)
- Excise the gel slice containing the DNA band with a clean, sharp scalpel. Minimize the size of the gel slice by removing excess polyacrylamide.
- Weigh the gel slice. Add 1–2 volumes of diffusion buffer to 1 volume of gel(i.e., 100–200 µl for each 100 mg of gel).
- Incubate at 50°C for 30 min.
- Centrifuge the sample for 1 min.
- Carefully remove the supernatant using a pipet or a drawn-out Pasteur pipet. Pass thesupernatant through a disposable plastic column or a syringe containing either aWhatman GF/C filter or packed, siliconized glass wool to remove any residualpolyacrylamide.
- Determine the volume of the recovered supernatant.
- Add 3 volumes of Buffer QG to 1 volume of supernatant and mix. Check that the col or of the mixture is yellow.
- Place a QIAquick Spin Column in a provided 2 ml collection tube.
- To bind DNA, apply the sample to the QIAquick Spin Column and centrifuge for30– 60 s.
- Discard flow-through and place QIAquick Spin Column back into the same collection tube.
- To wash, add 0.75 ml Buffer PE to column and centrifuge for 30–60 s.
- Discard flow-through and place QIAquick Spin Column back in the same tube. Centrifuge column for an additional 1 min at maximum speed.
- Place QIAquick Spin Column into a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of t he QIAquick Spin Column and centrifuge for 1 min.
VI. Plasmid Extraction:
Method:Plasmid Mini Kit I(200)
- Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for 12-16 hours at 37°C with vigorous shaking (300 rpm). Use a 10-20 mL culture tube or a flask with a volume of at least 4 times the volume of the culture.
- Centrifuge at 10,000 x g for 1 minute at room temperature. Decant or aspirate and discard the culture media.
- Add 250 µL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly. Complete resuspension of cell pellet is vital for obtaining good yields.
- Add 350 µL Solution III. Immediately invert several times until a flocculent white precipitate forms.
- Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
- Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
- Transfer the cleared supernatant from Step 5 by CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind® DNA Mini Column.
- Centrifuge at 10000g speed for 1 minute.
- Discard the filtrate and reuse the collection tube.
- Add 500 µL HB Buffer.
- Centrifuge at 10000g speed for 1 min.
- Discard the filtrate and reuse collection tube.
- Add 700 µL DNA Wash Buffer.
- Centrifuge at 10000g for 1 minute.
- Discard the filtrate and reuse the collection tube.
- Centrifuge the empty HiBind® DNA Mini Column for2 min at 13000g to dry the column matrix.
- Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
- Add 30-50 μL Elution Buffer or sterile deionized water directly to the center of the column membrane.
- Let sit at room temperature for 5 minute.
- Centrifuge at 13000g for 1 minute.
- Store DNA at -20°
VII. Standard PCR:
- Add DNA template up to 50ng into PCR tube,(x ul).
- Add reagents as follow:
- dNTP: 4ul
- 5x PSBuffer: 10ul
- Primer: 1ul (after diluted)
- Enzyme: 0.5ul
- ddH2O: (33.5-x)ul
Mix gently by vortex and briefly centrifuge to collect all components to the bottom of the tube.
- The amplification parameters will vary depending on the primers and the thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.
VIII. PCR purification：
- Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.
- Determine the volume of the PCR reaction. Transfer the sample into a clean 1.5ml microcentrifuge tube and add 4-5 volumes of Buffer CP. For PCR products smaller than 200bp, add 6 volumes of Buffer CP.
- Vortex thoroughly to mix. Briefly spin the tube to collect any drops from the inside of the lid.
- Place a HiBind DNA Mini Column into a provided 2 ml collection tube.
- Add the mixed sample from step 3 to the HiBind DNA Mini Column and centrifuge at 13,000 x g for 1 minute at room temperature. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
- Add 700µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
- Add 500µl of DNA Wash Buffer and centrifuge at 13,000 x g for 1 minute. Discard the flow-through liquid and place the HiBind DNA Mini Column back into the same collection tube.
- Centrifuge the empty HiBind DNA Mini column for 2 min at maximal speed ( ≥13,000 x g ) to dry the column matrix.
- Place the HiBind DNA Mini column into a clean 1.5ml microcentrifuge tube. Depending on the desire concentration of the final product, add 30-50 µl of Elution Buffer (10mM Tris, pH8.5) or water directly onto the center of column matrix.
- Transform ligation mix or other plasmid-containing reaction mixture into the desired bacterial strain, and incubate agar plates overnight at the appropriate temperature.
Set up 50 µl reactions as follows:
OneTaq Master Mix 25μl PCR primer 200 nM H2O to 50 µl
- Use a sterile toothpick to pick up individual colonies and dip into each reaction tube.
As soon as the solution looks cloudy, remove the toothpick. To create a stock of each individual colony
a.) Dip the toothpick into 3 ml growth media with appropriate antibiotics and culture overnight. or
b.) Streak the toothpick onto another agar plate containing the appropriate antibiotics and grow overnight.
Transfer reactions to a PCR cycler, and perform PCR following the guidelines below for cycling conditions:
- Load 4-6 µl of each PCR reaction directly onto an agarose gel, alongside an appropriate DNA ladder.
X. Agarose Gel Electrophoresis：
- Weigh agarose powder and TAE buffer and add them to a flask;
- Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave);
- Let the solution cool to about 40-50℃ and pour the solution into the gel casting tray with appreciate comb;
- Let the gel cool until it is solid;
- Carefully pull out the comb; Place the gel in the electrophoresis chamber;
- Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel;
- Pipette DNA samples mixed with appreciate amount of loading buffer and dye (GeneFinder) into wells on the gel;
- Run the gel at 120V for about 20 minutes;
XI. Extraction of E. coli genomic DNA:
- E. coli shake flask culture one day in advance
- 1.5 ml of the inoculum was placed in a centrifuge tube, centrifuged at 5000 rpm for
1 min, and the supernatant was discarded;
- Add 190ul DD water suspension precipitation, and add 10ul 10% SDS, shake until the solution becomes viscous;
- Add 1 ul of proteinase K (20 mg / ml), mix, incubate at 37 ° C for 1 hour;
- Add 30ul 5mol / L NaCl, mix;
- Add 30ul CTAB/NaCl solution, mix, heat at 65 ° C for 20min;
- Add 300 ul of phenol/chloroform/isoamyl alcohol extraction (25:24:1), centrifuge at 5000 rpm for 10 min;
- Take the supernatant, add 300 ul of chloroform / isoamyl alcohol (24:1), and centrifuge at 5000 rpm for 10 min;
- Take the supernatant, add 300 ul of isopropanol, mix by inversion, stand at room temperature for 10 min, precipitate the DNA, and centrifuge at 5000 rpm for 10 min;
- Discard the supernatant, add 500ul 70% ethanol to wash the pellet, and centrifuge at 5000rmp for 10min;
- Discard the supernatant and add 30 ul of DD water to dissolve the alcohol after it has evaporated.
- Dissolve in 20 ul TE and store at -80 °C.
XII. E.coli Competent cells:
- Innoculate a single colony of E. coli into 5 ml LB and grow O/N at 37
- Innoculate 1 ml into 100ml and grow to an O.D. 600 of 0.4
- Aliquot the culture into 2x 50ml pre-chilled Sorvall tubes and leave on ice for 5 -10 mins
- Centrifuge cells for 7 mins at 3000rpm, 4℃ without brakes
- Pour off supernatant and resuspend each pellet in 10 ml of ice-cold CaCl2 soln
- Centrifuge cells for 5 mins at 2500rpm, 4 degree. Discard supernatant and resuspend each pellet in 10 ml of cold CaCl2 solution.
- Keep resuspended cells on ice for 30min.
- Centrifuge cells for 5 mins at 2500rpm, 4℃. Discard supernatant and resuspend each pellet in 2ml of ice-cold CaCl2.
- Dispense cells into pre-chilled sterile eppendorfs.
- Freeze immediately @ -70 degresss
Notice: CaCl2 Soln: 60mM CaCl2, 15% Glycerol
XIII. Protein Extraction and expression:
- 80ul competent cells (BL21) + DNA samples (≤ 10ng; first add the plasmid and then add to small volume)
- Place on ice for 30 min → 42 ° C for 45 s → place on ice for 1~2 min → add 1 ml of LB-Ep tube → shake at 37 ° C for 1 h
- Take 100ul on LB resistant plate, overnight at 37 ° C
IPTG induced expression
- Pick colonies on the plate and draw 8 (for example) small squares.
- Pick up the bacteria with LB resistance solution, overnight at 37 ° C (OD test method: (Cuvette in A312, tube photometer in A317) Click cell growth---general test---smart test---cell growth (read-only) (wavelength is 600nm)--- measure Blanket (what is used as a blank for dilution, such a test should be blank with LB)---measure sample)
Cuvette liquid OD value, OD = A, diluted into Bml
(B * 0.2 / A = C, Bml LBK added Cml bacteria), OD = 0.2 (recommended to be diluted to 5ml or 10ml at the beginning ) Shake for 2h at 37℃, generally to OD = 0.5;
- Add IPTG induction generally optimally 0.5mM
- Initially induced expression requires exploration conditions: 1 IPTG amount: 0.1 ~ 1 mM gradient (0, 0.25, 0.5, 1)
- 4 hours(depend on the type of the protein) of culture at 37 degrees
- Centrifuge the cells to precipitate and wash them twice with PBS (make sure to wash) (add 3ml PBS) (move into the Ep tube when washing with PBS for the first time)
- lysate with mix: PMSF (1:40). protein inhibitor (1:100), DTT (500 mM → 5 mM); Add ulsate 500ul per tube; Ultrasonic disruption.
- Centrifuge at 44 ° C, 13000 rpm for 20 min, take the supernatant
XIV. List of some kits and reagents
|List of experimental kits||List of biological reagents|
|kits||origin||anti-Ha, anti-Flag antibody||Sigma,Abcam|
|Plasmid Mini Kit I(200)||Omega||anti-rabbit IgG||Proteintech|
|Plasmid Mini Kit(25)||Omega||anti-mouse IgG||Proteintech|
|Cycle-Pure Kit(200)||Omega||GoTaq Colonrless Master||Promega|
|Gel Extraction Kit I(200)||Omega||PrimerSTAR Max DNA Polymerase||Takara|
|Endo-free Plasmid Mini Kit II(50)||Omega||PrimerSTAR HS DNS Polymerase||Takara|
|GoScript Reverse Transcription System||Promega||DL2000 DNA Marker||Takara|
|QIAquick PCR Purification Kit(250)||QIAGEN||1Kb DNA Ladder(Dye Plus)||Takara|
|QIAquick Gel Extraction Kit I(250)||QIAGEN||6x DNA Loading Buffer||Takara|
|In-Fusion HD Cloning Kit||Takara||10x DNA Loading Buffer||Takara|
|DNA Ligation Kit Ver.2.1||Takara||Protease Inhibitor Cocktail||CWBIO|
|Blunting Kination Ligation Kit||Takara||2x Es Taq Master Mix||CWBIO|
|BCA Protein Assay Kit||CWBIO||Protease Inhibitor 8340||Sigma|
|cECL Western Blot Kit||CWBIO||PMSF||Sigma|
|ECL Western Blot Kit||WBIO||restained Protein Ladder||hermo|
|Plant RNA kit||OMEGA||Xma I, Restriction enzyme||NEB|
|Apa I, BamH I, EcoR I, , Kpn I, Not I, Sal I, Xba I, Spe I, Pst I, Xba I||Takara|