|NEB Q5® Hot Start High-Fidelity 2X Master Mix||25μL|
|step 1||step 2 (35cycles)||step 3||step 4|
|initial denaturation||denaturation||anneal||extension||final extension||stock|
2. Gel ElectrophoresisGel preparation
|Bio Rad TAE Buffer||30mL|
|Bio Rad Certified™ Molecular Biology Agarose||*|
|Invitrogen SYBR® Safe DNA gel stain||**|
- Sample Capture
- Weigh a DNase-free 1.5 ml microcentrifuge tube and record the weight.
- Using a clean scalpel, long wavelength (365 nm) ultraviolet light and minimal exposure time, cut out an agarose band containing the sample of interest. Place agarose gel band into a DNase-free 1.5 ml microcentrifuge tube (user supplied).
- Weigh the microcentrifuge tube plus agarose band and calculate the weight of the agarose slice.
- Add 10 μl Capture buffer type 3 for each 10 mg of gel slice, for example, add 300 μl Capture buffer type 3 to each 300 mg gel slice.
- Mix by inversion and incubate at 60°C for 15–30 minutes until the agarose is completely dissolved. Mix by inversion every 3 minutes.
- f. Once the agarose has completely dissolved check that the Capture buffer type 3-sample mix is yellow or pale orange in color.
- g. For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube.
- Sample Binding
- Centrifuge Capture buffer type 3- sample mix briefly to collect the liquid at the bottom of the tube.
- Transfer up to 800 μl Capture buffer type 3-sample mix onto the assembled GFX MicroSpin column and Collection tube.
- Incubate at room temperature for 1 minute.
- Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
- Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube.
- Repeat Sample Binding steps b. to e. As necessary until all sample is loaded.
- Wash & Dry
- Add 500 μl Wash buffer type 1 to the GFX MicroSpin column
- Spin the assembled column and Collection tube at 16 000 × g for 30 seconds.
- Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube (supplied by user).
- Add 10–50 μl Elution buffer type 4 OR type 6 to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.
- Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.
- Spin the assembled column and sample Collection tube at 16 000 × g for 1 minute to recover the purified DNA.
- Proceed to downstream application. Store the purified DNA at -20°C.
4. DNA Assembly
- Calculations Calculation rules: Vector:Insert=1:2 (in pmol)
- According to the calculations, mix the DNA samples and 10μL of NEBuilder® HiFi DNA Assembly Master Mix M5520AA.
- Put the mixture at 50ºC for 15 mins.
Total DNA amount: 0.03pmol~0.2pmol
Total DNA volume≤10μL
pmol = (ng×1000)/(bp×650)
5. Restriction Digest and DNA Ligation
- Restriction Digest
Restriction Enzyme 10 units DNA 1μg 10X NEBuffer *
- Incubate the above mixture at 37ºC for 4h.
- Conduct Gel electrophoresis and DNA and gel purification.
- DNA Ligation (20μL Reaction)
DNA Ligase 1μL DNA Ligase Reaction Buffer 2μL Vector 50ng Insert 37.5ng
6. Transformation of Escherichia coli
- Add the assembled product to 100μL of competent cells (Escherichia coli), place on ice for 20mins.
- Heat shock at 42ºC for 1min.
- Transfer to ice for 2mins.
- Add 900μL of LB to the tube, incubate at 37ºC for 1 hour. Shake at 250rpm.
- Warm the LB agar plate with antibiotics* to 37ºC.
- Centrifuge at 12000rpm for 30s. Concentrate the cells to 100μL.
- Spread 100μL cells onto the plate.
- Incubate overnight at 37ºC.
7. Miniprep Plasmid Purification
- Pellet 1.5mL bacteria each tube and centrifuge at 6800ˣg for 3mins at room temperature for 3 times.
- Resuspend pelleted bacteria in 250mL Buffer P1.
- Add 250mL Buffer P2 and mix 6 times.
- Add 350mL Buffer N3 and mix 6 times.
- Centrifuge for 10mins at 13000rpm.
- Apply 800mL supernatant to the column, centrifuge for 30s.
- Add 0.5mL Buffer PB and centrifuge for 30s.
8. Exfection™ Plasmid EF midi
- Pellet 50~100 ml of bacterial culture by centrifugation for 5 min at 10,000 x g in a tabletop centrifuge. Discard the supernatant as much as possible.
- Bacterial culture grows for 16 to 24 hours in LB-broth containing selective antibiotics. Resuspend pelleted bacterial cells thoroughly in 4 ml of buffer P1.
- It is essential to thoroughly resuspend the cell pellet. * Add RNase A before first use of buffer P1.
- Add 4 ml of buffer P2 and mix by inverting the tube 5~6 times (DO NOT VORTEX). Incubate until the cell suspension becomes clear and viscous, but DO NOT incubate for more than 5 min.
- Add 4 ml of buffer P3 and thoroughly but gently mix by inverting the tube 7~8 times (DO NOT VORTEX).
- Mix the solution gently but completely and immediately after the addition of buffer P3 for optimal precipitation.
- Pour all of the lysate into EzClearTM filter (blue ring) sitting on a 50 ml conical collection tube (provided). Let it stand for 2 min and centrifuge for 2 min at 1,500 x g (2,800 rpm).
- Apply 500 ul of buffer ER to the filtrate and close the cap of 50 ml conical tube.
- Vortex to mix and incubate for 15 min on ice.
- Incubate for 15 min at 37˚C and centrifuge for 2 min at 1,500 x g (2,800 rpm).
- Transfer carefully the upper phase (clear) into a fresh 15 ml conical tube (provided) by pipetting.
- Add 1/2 volume of buffer EG to the transfer and invert several times to mix completely.
- Transfer all of the mixture to SV Midi column (clear ring) by decanting or pipetting. Centrifuge for 2 min at 1,500 x g (2,800 rpm). Remove the column, discard the pass-through, and re-insert the column to the collection tube.
- Apply 10 ml of buffer EW1 and centrifuge for 2 min at 1,500 x g (2,800 rpm). Remove the column, discard the pass-through, and re-insert the column to the collection tube.
- Apply 10 ml of buffer EW2 and centrifuge for 2 min at 1,500 x g (2,800 rpm). Remove the column, discard the pass-through, and re-insert the column to the collection tube.
- Apply 3 ml of buffer EW2 and centrifuge for 15 min at 4,500 x g (5,000 rpm). Transfer the column to a new 50 ml conical tube (provided).
- Add 0.6 ml of buffer EF or endotoxin-free water directly onto the center of the column membrane and close the cap. Incubate for 5 min at room temperature and centrifuge for 5 min at 4,500 x g (5,000 rpm).
- Wash cell pellet with PBS.
- Resuspend cell pellet with 100ul lysis buffer and set on ice for 10 min.
- Sonication: use microtip, set 40% AMPL, 45 secs processing, 1 sec on-off, sonicate for 3 times.
- Centrifuge at max speed for 20 min.
- Collect the supernatant for soluble fraction
- Resuspend the precipitate with 100ul urea buffer
10.SDS-PAGE preparationgel preparation (2 gels)
10% Resolving Gel:
|1.5M Tris (pH8.8)||4ml|
- Add resolving gel solution to the glass plates until the solution reaches the level of the fill line.
- Carefully overlay the resolving gel solution with butanol, or isopropanol.
- Allow the resolving gel to polymerize (5–10 min). The interface becomes more distinct as the gel polymerizes.
- Verify polymerization by examining left over acrylamide in the tube.
- If overlay was used, pour off the overlay solution and rinse with water (dispose of waste in the appropriate manner). Wick out any remaining liquid with a piece of blotting paper, and proceed to Prepare stacking gel solution.
|0.5M Tris (pH6.8)||1.1ml|
|Water 40ul 10% SDS||2.5ml|
- Add the stacking gel solution until it reaches the upper edge of the front glass plate.
- Insert the comb slowly by starting at one end and sliding it between the glass plates until both ends are in place.
- Allow the stacking gel to polymerize (5–10 min).
- Load protein ladder and samples to the gel.
- Run the gel at 60V for 1 hour and increase to 120V for another one hour.
12.Coomassie blue stain
- Wash the gel with water for 10 min on a shaker.
- Soak the gel in Coomassie Brilliant Blue and incubate at room temp. for an hour on a shaker.
- After the gel is a uniform blue color, wash the gel 3 times with water (5 min each time).
- Destain the gel with water overnight on a shaker.
13.Western BlotGel transfer
- Prepare 1x transfer buffer (50mL 5x transfer buffer + 150mL MilliQ water + 50mL Ethanol).
- Transfer the SDS gel into wet transfer buffer.
- Soak the membrane and transfer stack in the wet transfer buffer.
- Cassette assembly:
- Place one transfer stack on the cassette.
- Place the membrane on the transfer stack.
- Place the gel on the membrane.
- Place another layer of transfer stack on the gel.
- Close the cassette firmly and put into A or B of the semi-dry transfer machine.
- Run the machine by choosing option: 1.5mm, 10 minutes
- Block the membrane in blocking buffer (1g non-fat dry milk + 20mL TBST) for an hour on a shaker .
- Dilute primary antibody in blocking buffer (1:2000 dilution).
- Incubate the membrane in diluted primary antibody overnight at 4 degree.
14. IPTG induction
- From a relatively fresh plate (<4 weeks) pick a colony and grow O/N at 37C) in 2ml LB+AMP in a 15ml snap cap tube in a shaker.
- Dilute 1:50 in 4ml LB+AMP and grow 3 hours at 37 C in 15ml snap cap tube in a shaker.
- Prepare 1ml LB+AMP+5mM IPTG and 1ml LB+AMP+2mM IPTG in tubes and prewarm to 37 C about 10min before use.
- Transfer 1ml from the culture and spin at 14,000rpm for 30s at RT. Remove supernatant. Freeze pellet at -20 until needed. This is the uninduced control.
- After 3 hours of incubation, add 1 ml LB+AMP+5mM IPTG and 1ml LB+AMP+2mM IPTG to 4ml to make 1mM IPTG and 0.4mM IPTG.
- Transfer 1 ml from induced sample and spin at 14,000rpm for 30s at RT after incubation of 0.5h, 1h, 2h and 4h. Remove supernatant. Freeze pellet at -20 until needed.
- Use microtip.
- Set 40% AMPL.
- 45 secs processing.
- 1 sec on-off, sonicate for 3 times.
- Add 1500u/ml tyrosinase to the cell lysate to get 333u/ml.
- Incubate at 25C for 1 hour.
- Soak the cloth and incubate at 4C overnight.
16. Vertical fire test
- The solution was made in a beaker and the bedsheets was soaked in the beaker for a day to ensure complete absorption.
- The bedsheets were taken out and put on the lab racks
- The equipment made by ourselves was set up
- The bedsheets were hold using four clips and hole by the lab racks
- The bedsheets were ignited for 5 seconds and put out
- Step 4 and 5 were repeated for every soaked bedsheet
17. Wood test -- Performed by IDQ
18. Adhesion test
- Soak the cloth with the protein mixture (protein with RFP) and incubate at 4C overnight
- Observe the result through Nikon A1MP + fluorescence confocal microscope (Nikon, Japan) before soaking and washing
- Rinse the cloth and wash the cloth by water
- Observe the result through Nikon A1MP + fluorescence confocal microscope (Nikon, Japan) again to check the adhesiveness of the protein