Team:XHD-ShanDong-China/Protocol

Protocols

MG1655 Anabiosis and CP Plasmid process

MG1655	Cp plasmid
Melt the glycerin bacteria on the ice	Melt the glycerin bacteria on the ice
Add 5ml liquid LB	Mark the 12 ml shake bacteria tube
Add 100ul MG1655 glycerin bacteria liquid	Add 5ul 50mg/ml Cm antibiotic
Put it in the table	Add 100ul MG1655 glycerin bacteria liquid
Culture overnight	Put it in the table
	Culture overnight

PCR Process (one cycle)

1.Predenaturing:98℃continues 3mins——Strands Separate
2.Denaturing:98℃ continues 10s——Strands Separate
3.Annealing：55℃ continues 10s——primers bind template.
4.Extnsion:72℃ continues 25s——synthesise new strand
5.Go to step2×30
6. Extnsion:72℃ continues 2 min
7.4℃∞

Plasmid extraction（OMEGA kit）

1)In a 15mL sterile tube, inoculate the Escherichia coli carrying the required plasmid into 5mL LB medium, and shake culture for 12-16h
2)Pellet 1.5-5.0mL bacteria by centrifugation at 10000 x g for 1 min at room temperature
3)Resuspend the bacterial pellet by adding 250uL of Solution I/RNaseA and vortexing
4)Add 250uL of Solution II and getly mix by inverting and rotating the tube several times to obtain a clear lysate
5)Add 350uL of Solution III and mix immediately by inverting the tube several times until a flocculent white precipitate forms
6)Centrifuge at 10000 x g for 10 minutes at room temperature
7)Add the cleared supernatant by CAREFULLY aspirating it into a clean HiBind Miniprep Column(I) assembled in a provided 2mL collection tube. Centrifuge for 1min at 10000 x g at room temperature to completely pass lysate through the HiBind Miniprep Column(I)and discard flow-through liquid
8)Add 500uL of Buffer HB, Centrifuge for 1 min at 10000 x g at room temperature, discard flow-through liquid
9)Add 700uL of DNA Wash Buffer diluted with absolute ethanol, centrifuge for 1min at 10000 x g at room temperature, discard flow-through liquid
10)repeat step 9
11)Centrifuge the empty column for 2min at 13000 x g
12)Place the column into a clean 1.5mL microcentrifuge tube. Add 30uL to 50uL of Elution Buffer or sterile deionized water directly onto the column matrix and let it sit at room temperature for 1~2 minutes. Centrifuge for 1 min at 13000 x g to elute DNA
13)Yield and quality of the plasmid solution in a 1.5mL centrifuge tube and store it.

Plasmid mini purification protocol

1.Resuspend the bacterial pellet in 0.2ml(200ul) of Buffer S1 to mix thoroughly, then pipette the suspension inti a 2ml microcentrifuge tube.(ensure that RNA se A has been added to Buffer P1.The bacteria should resuspended completely, leaving no cell clumps.h
2.Add 0.2 ml (200 m l) of Solution 2 , Mix gently, and incubate at room temperature for 5 min. After addition of Solution 2, the solution should be mixed gently, but thoroughly, by inverting the tube A-6 times. Do not vortex, as this will result in shearing of genomic DNA .The lysate should appear viscous. Do not allow the lysis reaction to pro for more than 5 min. After use, the bottle containing solution2 should be closed immediately to avoid any reaction between the NaOH and C02 in the air. If the buffer is left open for any length of time, it should be prepared fresh from stock solutions.
3.Add 0.2 ml (200 m l) of chilled Solution 3, mix immediately but gently. After addition of Solusion3, the solution becomes cloudy and very viscous. To avoid localized potassium dodecyl sulfate precipitation, mix the solution gently, butthoroughly, immediately after addition of Solution3.Mix by inverting the tube 4-6 times.
4.Centrifuge at maximum speed in a microfuge for 5 min. Do step 5 during the centrifugation. Before loading the centrifuge, the sample should be mixed again. Centrifugation should be performed at maximum speed in 1.5-ml or 2-ml microfuge tubes (e.g. 10,000-13,000 rpm in a microfuge).Maximum speed corresponds to 14,000-18,000 x g for most microfuges.
5.Insert the adsorption column into microcentrifuge tube, fetch the supernate in step4, add the adsorption, and then centrifuge at 12000rpm for 1min.
6.Abandon the filtrate, add 700uL WB in the adsorption, and then centrifuge at 12000rpm for 1min, abandon the filtrate, repeat the process again.
7.Abandon the filtrate, centrifuge at 12000rpm for 1min, and then abandon the filtrate.
8.Put the adsorption in to a new 1.5 ml microcentrifuge tube（mark it）, add 70ul ddH2O, after 1minutes’ standing, 12000rpm centrifuge at 12000rpm for 1min, keep the plasmid that in the 1.5ml microcentrifuge.

Electrophoresis Gel (simgen)

1)Cut out the agarose gel containing the target DNA fragment under UV light, weigh the gel and record it as A mg, and put it in a 2mL centrifuge tube
2)Add 3A uL of Buffer G, 50℃ water bath until the gel is completely dissolved
3)Transfer the liquid of step 2 to a nucleic acid purification column (the purification column is placed in a 2mL centrifuge tube), centrifuge at 12000rpm for 30s, and discard the filtrate
4)Add 500uL Buffer WS, centrifuge at 12000rpm for 30s, discard flow-through liquid
5)Add 700uL Buffer WG, centrifuge at 12000rpm for 30s, discard flow-through liquid
6)Repeat step 5
7)Centrifuge at 14000rpm for 1 min, discard 2mL centrifuge tube
8)Put the nucleic acid purification column into a new 1.5mL centrifuge tube, add 25~30uL Buffer TE to the center of the purification column, let it stand at room temperature for 1min, and centrifuge at 12000rpm for 30s
9)Yield and quality of the DNA solution in a 1.5mL centrifuge tube and store it.

Gibson Assembly Protocol

1.set up the following reaction on ice:

	Recommended amount of fragment Used for Assembly
	2-3 fragment Assembly	4-6 Fragment Assembly	Positive Control
Total Amount of Fragments	0.02-0.5 pmols* X μl	0.2–1 pmols* X μl	10μl
Gibson Assembly Master Mix(2X)	10μl	10μl	10μl
Deionized H2O	10-Xμl	10-Xμl	0
Total Volume	20 μl***	20 μl***	20μl

* Control reagents are provided for 5 experiments.
** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
2.Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation For thermocycler (placement in middle is better) turn it on, go to saved programs, enter 330, and then run.
3.Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

Gibson Assembly Chemical Transformation Protocol

1.Thaw competent cells on ice.
2.Add 2 μl of the chilled assembly product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
3.Place the mixture on ice for 30 minutes. Do not mix.
4.Heat shock at 42°C for 30 seconds. Do not mix.
5.Transfer tubes to ice for 2 minutes.
6.Add 950 μl of room-temperature SOC media to the tube. (*YPD media for yeast).
7.Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8.Warm selection plates to 37°C.
8.Warm selection plates to 37°C.
9.Spread 100 μl of the cells onto the selection plates. Use Amp plates for positive control sample. (*chloramphenicol -camr plates in our case).
10.Incubate overnight at 37°C.