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.
all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (95°C).
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
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
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.
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
.Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended.
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.
Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until colour is uniformly yellow and a precipitate forms.Incubate for 2 minutes.
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.
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.
Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
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
Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
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.
If monitoring cell density by OD600, record final OD readings prior to harvesting.
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.
Store the pellet at -20°C or -80°C or process immediately.
Resuspend the cell pellet in NEBExpress E. coli Lysis Reagent by pipetting or vortexing briefly until the suspension is homogenous:
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.
If harvested cells are weighed, use 5 mL of NEBExpress E. coli Lysis Reagent per 1 gram of cells.
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.
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).
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.
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.
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.
1.Since the protein produced in BL21 matches the estimated kDa of our fusion protein, the plasmid insertion is proved to be successful, and our desired protein
Gel electrophoresis is a laboratory method used to seperate mixtures of DNA, RNA, or proteins according to molecular size. In gel electrophoresis, the molecules to be seperated are pushed by an electric field through a gel that contains small pores
Preparation of Agarose Gel
Dilute 50X TAE Buffer to 1X TAE buffer.
Add desired amount of ultra-pure agarose to 1X TAE buffer in a flask.
Microwave solution for 1 min. Microwave for longer if there is undissolved agarose.
Allow the solution to cool for 1 min.
Pour the solution inro the gel tray. Insert the comb into the top of the gel.
after 15 mins, 3μL GelGreen® Nucleic Acid Gel Stain was added into the gel solution.
Running gel electrophoresis
Once the gel has solidified, carefully remove the comb by pulling straight up.
Prepare the samples by adding 6X loading buffer to each.
Load 15μL of DNA ladder into one of the lane.
Load samples into wells. Avoid bubbles.
Remove the tray with the gel and image with UV
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.
Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear.
Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture.
Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 minutes. Do not mix.
Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
Place on ice for 5 minutes. Do not mix.
Pipette 950 µl of room temperature SOC into the mixture.
Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
Warm selection plates to 37°C.
Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.
Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
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. I
ncubate 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.
Place the the gel in the SDS-PAGE stand
Place the stand with the gel in the SDS-PAGE apparatus bath
fill the space between the gels with running buffer
Take off the combs of the gel (take it out gently)
Load the sample to each well
Do not use the wells at the left and right end of the gel
fill the SDS-PAGE apparatus bath with running buffer to the bottom of the gel
Place the cover of the SDS-PAGE
Plug the cables to the SDS-PAGE apparatus power source
Turn of the SDS-PAGE power source
Set to constant voltage
Use 200
Press the Run button to start the electrophoresis
The voltage increases to 200 V from 0 V
For 4% staking gel and 12.5 % separating gel the current reaches 50-60 mA and the power 10 -12 W
The current the the power decreases in time but not reach zero (if it reaches zero see the troubleshoot)
For the state type of gel it takes 35 - 40 min