Golden Gate Assembly Kit

The NEBridge Golden Gate Assembly Kit (BsaI-HFv2) contains an optimized mix of BsaI-HFv2 and T4 DNA Ligase. Together these enzymes can direct the assembly of multiple inserts/modules using the Golden Gate approach. Also included is the pGGAselect destination plasmid, which provides a backbone for your assembly, features convenient restriction enzyme sites for subcloning, and has T7/SP6 promoter sequences to enable in vitro transcription.

Reagent	Negative control	Assenbly reaction
pGGAselect Destionation Plasmid*, 75 ng/μl	1 μl	1 μl
Inserts (user provided):- if precloned**- if in amplicon form***	1 μl	75 ng each plasmid 2:1 molar ratio (insert : vector; pGGAselect = 2155 bp; 75 ng = 0.05 pmol)
T4 DNA Ligase Buffer + peg 3350 (10X)	2 μl	2 μl
NEB Golden Gate Assembly Mix	1 - 2 μl****	1 - 2 μl
Nuclease-free H2O	to 20 μl	to 20 μl

** Precloned inserts must possess BsaI restriction sites at both ends of the insert sequence and in the proper orientation.
*** Amplicon inserts must possess 5´ flanking bases and BsaI restriction sites at both ends of the amplicon and in the proper orientation.
**** For assemblies < 10 inserts, use 1 μl : for assemblies > 10 inserts, use 2 μl.

Inset number	Suggested Assembly Protocol
For 1 Insert	37°C, 5 min (cloning) or 37°, 1 hr (library preparation) → 60°C, 5 min
For 2-4 Inserts	37°C, 1 hr → 60°C, 5 min
For 5-10 Inserts	(37°C, 1 min → 16°C, 1 min) x 30 → 60°C, 5 min
For 11 - 20+ inserts	(37°C, 5 min → 16°C, 5 min) x 30 → 60°C, 5 min

Master mix

water(2x stuff μl) -> ligase buffer -> gene -> bsaI (immediately put back in freezer(-20)) -> t4 ligase -> plasmids -> label tubes -> flick -> centrifuge -> thermal cycler(annealing) 1 hr (short) / 3 hrs (long) 37°C

The Polymerase Chain Reaction (PCR) is a powerful and sensitive technique for DNA amplification (1). Taq DNA Polymerase is an enzyme widely used in PCR (2). The following guidelines are provided to ensure successful PCR using New England Biolabs’ Taq 2X Master Mix. These guidelines cover routine PCR. Amplification of templates with high GC content, high secondary structure, low template concentrations, or amplicons greater than 5 kb may require further optimization.

Reaction setup

all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).

Component	25 µl reaction	50 µl reaction	Final Conc.
10 µM Forward Primer	0.5 μl	1 μl	0.2 µM (0.05–1 µM)
10 µM Reverse Primer	0.5μl	1 μl	0.2 µM (0.05–1 µM)
Template DNA	variable	variable	<1,000 ng
Taq 2X Master Mix	12.5μl	25 μl	1X
Nuclease-free water	to 25 µl	to 50 µl

Transfer PCR tubes from ice to a PCR machine with the block preheated to 95°C and begin thermocycling

Thermocycling conditions for a routine PCR:

STEP	TEMP	TIME
Initial Denaturation	95°C	30 seconds
30 Cycles	95°C 45-68°C 68°C	15-30 seconds 15-60 seconds 1 minute per kb
Final Extension	68°C	5 minutes
Hold	4-10°C

Plasmid Miniprep Kit

The Plasmid Miniprep Kit is a rapid and reliable method for the purification of high quality plasmid DNA. This method employs standard cell resuspension, alkaline lysis, and neutralization steps, with the additional benefit of color indicators at certain steps to easily monitor completion. Unique wash buffers ensure salts, proteins, RNA and other cellular components are removed, allowing low-volume elution of concentrated, highly pure DNA. Elution in as little as 30 μl provides concentrated DNA for use in downstream applications, such as restriction digests, DNA sequencing, PCR and other enzymatic manipulations.

PLASMID	REPLICON	COPY NUMBER	CLASSIFICATION
pUC and its derivatives	pMB1	>75	High copy
pBR322 and its derivatives	pMB1	15–20	Low copy
pACYC and its derivatives	p15A	10–12	Low copy
pSC101	pSC101	-5

Buffer Preparation:

Add ethanol to Monarch Plasmid Wash Buffer 2 prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2).

• For 50-prep kit add 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2
• For 250-prep kit add 120 ml of ethanol to 30 ml of Monarch Plasmid Wash Buffer 2

Protocol:

All centrifugation steps should be carried out at 16,000 x g

1.Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended.

2.Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Incubate for one minute.

3.Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms.Incubate for 2 minutes.

4.Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.

Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.

5.Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute.

6.Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

7.Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through.

8.Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

E. coli Lysis Reagent

NEBExpress E. coli Lysis Reagent is a chemical lysis solution composed of a proprietary mix of non-ionic and zwitterionic detergents and Tris-based buffer. It allows disruption of E. coli cells without denaturing soluble proteins.

Protocol

1.If monitoring cell density by OD600, record final OD readings prior to harvesting.

2.Harvest cells by centrifugation at 16,000 x g for 10 minutes. For larger volumes, centrifuge for ≥30 minutes especially if at ≤10,000 x g. Discard the medium and, if necessary, weigh the wet cell pellet.

3.Store the pellet at -20°C or -80°C or process immediately.

4.Resuspend the cell pellet in NEBExpress E. coli Lysis Reagent by pipetting or vortexing briefly until the suspension is homogenous:

5.Use 0.025 - 0.075 mL of NEBExpress E. coli Lysis Reagent for every 1 UOD600 harvested. To calculate the UOD600, multiply the volume harvested by the OD600 reading.
For example, a 5 mL culture harvested at OD600 1 gives 5 mL x 1.0 = 5 UOD600. In this example, 0.125 – 0.375 mL Lysis Reagent is required to lyse efficiently.

6.If harvested cells are weighed, use 5 mL of NEBExpress E. coli Lysis Reagent per 1 gram of cells.

7.Incubate the resuspended cells at room temperature for 10 - 20 min with gentle shaking, gentle rotation, or swirling. Lysis is usually visible with a clearance of the suspension.

8.Centrifuge the lysate at 16,000 x g for 10 min at 4°C to pellet the insoluble material and cell debris (30 min or longer for large volumes and lower speed).

9.Carefully transfer the supernatant into a sterile container for analysis or purification. This soluble fraction can be stored at 4°C for a few hours or -20°C or -80°C for longer term storage.

10.If needed, resuspend the insoluble pellet in 50 mM Tris-HCl pH 7.5 or any desired buffer for analysis or purification of inclusion bodies.

Ni Spin Columns

Spin columns containing an affinity matrix for the small-scale isolation and purification of polyhistidine-tagged (His-tagged) fusion proteins. Immobilized Metal Affinity Chromatography (IMAC) purifications employing NEBExpress® Ni Spin columns can be performed under native or denaturing conditions, yielding highly pure target in a single protein purification step. This enables screening of expression conditions and streamlines the functional and structural characterization of the target protein.

Protocol

1.Remove the bottom tab of the column and place the column in a collection tube, loosen the cap.

2.Centrifuge column at 800 x g for 1 minute, discard the buffer.

3.Add 250 μl of Lysis/Binding buffer to the column.

4.Centrifuge column at 800 x g for 1 minute, discard the Lysis/Binding buffer.

5.Add up to 500 μl of sample lysate to the column, tap to mix and allow binding for 2 minutes.

6.Centrifuge column at 800 x g for 1 minute, retain flow through.

7.Place the column in a new 2 ml microcentrifuge tube.

8.Add 250 μl of Wash buffer to the column and centrifuge at 800 x g for 1 minute, repeat twice. Retain the washes.

9.Add 200 μl of Elution buffer to the column. Tap the column repeatedly to mix.

10.Centrifuge at 800 x g for 1 minute, retain the eluate.

11.Repeat elution step once, collecting fraction in a new microcentrifuge tube.

Gel elctrophoresis

Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.

Protocol

Preparation of Agarose Gel

1. Dilute 50X TAE Buffer to 1X TAE buffer.
2. Add desired amount of ultra-pure agarose to 1X TAE buffer in a flask.
3. Microwave solution for 1 min. Microwave for longer if there is undissolved agarose. Allow the solution to cool for 1 min.
4. Pour the solution inro the gel tray. Insert the comb into the top of the gel. After 15mins, 3μL GelGreen® Nucleic Acid Gel Stain was added into the gel solution. Allow the gel to solidify another 15mins.

Running gel electrophoresis

1. Once the gel has solidified, carefully remove the comb by pulling straight up.
2. Prepare the samples by adding 6X loading buffer to each.
3. Load 15μL of DNA ladder into one of the lane.
4. Load samples into wells. Avoid bubbles.
5. Remove the tray with the gel and image with UV

Liposome preparation

Thin-Film hydration followed by extrusion method for liposome preparation involves making a thin lipid film in a round-bottom flask by the removal of organic solvent. Upon the addition and agitation of the dispersion medium, heterogeneous liposomes are formed. Finally, after extrusion through polycarbonate membranes, homogeneous small liposomes are obtained.

Protocol

Preparation of the Thin Film

1. Add 5 mL of chloroform to rinse a 50-mL glass round-bottom fl ask. The round-bottom fl ask may be used immediately after rinsing.
2. To prepare 2 mL of 10 mg/mL PC/cholesterol liposomes (1:1, molar ratio), add 670 μL of 10 mg/mL PC stock solution and 1330 μL of 10 mg/mL cholesterolstock solution and mix them briefl y in a chemical hood. Then evaporate the organic solvent on a rotary evaporator. The fl asks may be dipped in a warm water bath, usually 40–45 °C, to make the evaporation process faster.
3. Put the fl ask in a vacuum desiccator overnight to remove organic solvent residue. Cover the opening of the fl ask by a piece of stretched parafi lm to prevent the entry of dusts or contaminants and poke some small holes in the parafi lm by a needle.

Hydration of the Thin Film

1. Add 2 mL of PBS to the dry lipid fi lm.
2. Vortex the solution for 10 s on maximum speed three times to suspend the lipid materials in the solution. Some lipid materials on the edge of the dry lipid fi lm may stay on the fl ask and form a ring. In this case, sonicate the lipids in a water bath sonicator for 15 s to help the lipids suspend in the solution.
3. Once all lipid materials are suspended in the solution, allow the suspension to stand at 4 °C overnight to effi ciently hydrate the lipid materials.

Extrusion

1. Sonicate the liposome preparation in a water bath for 10–20 min to remove all visible precipitates. The obtained milky crude liposome sample contains mostly MLVs . The particle size will decrease with prolonged sonicationtime.
2. Put the solution into a syringe filter with the pore size of 400 nm .
3. Extrude the liposome preparation by passing the suspension through the syringe filter. The sample will look semi-translucent. It is lipsome.
4. Store the liposome sample at 4 °C.

High Efficiency Transformation using both in DH5-alpha and BL21(DE3)

In transformation, cell are made competent (able to take up exogenous DNA) by treatment with divalent cations such as calcium chloride, which make the bacterial cell wall more permeable to DNA. Heat shock is used to temporarily form pores in the cell membrane, allowing transfer of the exogenous DNA into the cell.

Protocol

1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear.
2. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
3. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture.
4. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
5. Place the mixture on ice for 30 minutes. Do not mix.
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
7. Place on ice for 5 minutes. Do not mix.
8. Pipette 950 µl of room temperature SOC into the mixture.
9. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
10. Warm selection plates to 37°C.
11. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
12. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
13. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

Protocol

Transform expression plasmid into BL21(DE3). Plate on antibiotic selection plates and incubate overnight at 37°C. Resuspend a single colony in 10 ml liquid culture with antibiotic. Incubate at 37°C until OD600 reaches 0.4–0.8. Induce with 4 or 40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) and induce for 3 to 5 hours at 37°C. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only.

Protocol

1. 1.Place the the gel in the SDS-PAGE stand
2. 2.Place the stand with the gel in the SDS-PAGE apparatus bath
3. 3.fill the space between the gels with running buffer
4. 4.Take off the combs of the gel (take it out gently)
5. 5.Load the sample to each well
6. 6.Do not use the wells at the left and right end of the gel
7. 7.fill the SDS-PAGE apparatus bath with running buffer to the bottom of the gel
8. 8.Place the cover of the SDS-PAGE
9. 9.Plug the cables to the SDS-PAGE apparatus power source
10. 10.Turn of the SDS-PAGE power source
11. 11.Set to constant voltage
12. 12.Use 200 V
13. 13.Press the Run button to start the electrophoresis
        1.The voltage increases to 200 V from 0 V
        2.For 4% staking gel and 12.5 % separating gel the current reaches 50-60 mA and the power 10 -12 W
        3.The current the the power decreases in time but not reach zero (if it reaches zero see the troubleshoot)
        4.For the state type of gel it takes 35 - 40 min

Protocol

Use ultrasonic bath to insert the GFP into our liposome.