*depends on the desired gel concentration
**1μL SYBR® Safe per 1mL TAE Buffer
Sample loading
DNA sample : Gel Loading Dye Purple (6x) = 5:1.
3.Gel purification
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).
Elution
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)
Total DNA amount: 0.03pmol~0.2pmol
Total DNA volume≤10μL
pmol = (ng×1000)/(bp×650)
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.
5. Restriction Digest and DNA Ligation
Restriction Digest
Restriction Enzyme
10 units
DNA
1μg
10X NEBuffer
*
*depends on total volume
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
Incubate the above mixture at 50°C for 20mins.
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.
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).
9.Protein Extraction
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 preparation
gel preparation (2 gels)
10% Resolving Gel:
40% Acrylamide
4ml
1.5M Tris (pH8.8)
4ml
water
7.8ml
10% SDS
160µl
10% APS
80µl
TEMED
8µl
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.
4% Stacking gel:
40% Acrylamide
0.4ml
0.5M Tris (pH6.8)
1.1ml
Water 40ul 10% SDS
2.5ml
10% APS
20µl
TEMED
4µl
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).
Release the clamp and remove the plate. The gel can be used immediately, or wrapped in a damp paper towel and stored at 4°C for future use.
11.Running Gel
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 Blot
Gel 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
Immunoblotting
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.
15. Sonication
Use microtip.
Set 40% AMPL.
45 secs processing.
1 sec on-off, sonicate for 3 times.
Adhesion the protein to cloth
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
Bs476-part4:1970 tested was used to test fire retardancy of wood. It is a test on build materials and structures. It is a Non-combustibility test for materials.
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
