Plasmid preparation and Restriction digestion

Exp 1 Plasmid mini-preparation from E. coli

Plasmids: small, extrachromosomal circular molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells. contain an origin of replication and replicate independently Usually carry a few genes, one of which may confer resistance to antibacterial substance. Contain MCS.

Plasmids Minipreparation (miniprep) Plasmids are resistant to chemical denaturation, thus can be isolated from the chromosomal DNA easily with method of alkaline lysis. Isolation of plasmid DNA from a few milliliters (mL) of bacterial culture

Grow the cell Harvest the cell by centrifugation Resuspend the cell pellet Alkaline lysis of the cell neutralization Ethanol precipitation

Mid-log bacteria supernatant pellet Plasmid DNA Solution I extraction drying Sol:II lysis precipitation Plasmid DNA Sol III neutralization washing

Reagents LB medium SolutionⅠ Solution Ⅱ (fresh) Solution III ethanol, 70% ethanol

Fast Plasmid Prep Procedure 100 1. Pellet 1.5 ml of fresh bacterial culture at 12000 rpm for 1 minute in a 1.5 ml microfuge tube. 2. Remove medium by decanting, taking care not to disturb bacterial pellet.

Fast Plasmid Prep Procedure 3. Add 100 µl solution I and suspend the pellet 4. Add 200 µl solution II and mix the content gently, leave it on ice for 3 min 5. Add 150 µl solution III and mix the content gently, leave it on ice for 3 min

Fast Plasmid Prep Procedure 7.Transfer supernatant to a 1.5ml tube 6. Centrifuge at 12000rpm for 10min.

Fast Plasmid Prep Procedure 9. Centrifuge at 12000rpm for 10min. 8.Add 2 volume of ethanol 10. Remove and discard supernatant. The pellet is the precipitated plasmids 11.Wash the pellet with 70% ethanol, Centrifuge at 12000rpm for 1min, discard supernatant. Dry the pellet by inverting the tube and leave it at R.T for 10min 12. Resuspend the pellet in 20μl DDW, add 1μl RNase.

Restriction Enzymes digestion and agarose gel electrophoresis

Restriction endonuclease Bacterial enzymes which cut DNA into defined and reproducible fragments 2. Key discovery which allowed the DNA cloning to become a reality

Recognition sequences Recognize 4-8 bp. Most recognition sequences are 6 bp. Highly specific Restriction enzymes Commercially available Require Mg2+ for enzymatic activity

Restriction enzymes create DNAs with “blunt” or “sticky” ends AAGCTT TTCGAA 5’ 3’ 5’ HindIII 3’ CCCGGG GGGCCC 5’ 3’ 3’ 5’ SmaI GGTACC CCATGG 5’ 3’ 3’ 5’ KpnI

Reaction mixture volume（µL） H2O 5 10×Buffer K 1 Plasmid DNA 3 BamH I total 10 volume（µL） H2O 10 10×Buffer K 2 Plasmids DNA 6 Bam HI Xho I 1 for each total 20

Agrose Gel Electrophoresis

Agarose Gel Electrophoresis Agarose gel electrophoresis is routinely used for the analysis of DNA. Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. We will be using agarose gel electrophoresis to determine the presence and size of plasmid.

+ - DNA H  O2  Power • DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positive pole (anode). H  O2 • An agarose gel is used to slow the movement of DNA and separate by size. + - Power Scanning Electron Micrograph of Agarose Gel (1×1 µm)  • Polymerized agarose is porous, allowing for the movement of DNA

+ - How fast will the DNA migrate? DNA small large Power strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size DNA + - Power small large

REAGENTS AND APPARATUS Agarose Buffer (running buffer and sample buffer) Size marker EB Casting tray Comb Electrophoresis apparatus (gel tank) Cover Power supply UV light

Agarose is a linear polymer extracted from seaweed. D-galactose 3,6-anhydro L-galactose Sweetened agarose gels have been eaten in the Far East since the 17th century. Agarose was first used in biology as a culture medium for Tuberculosis bacteria in 1882 Agarose is a linear polymer extracted from seaweed.

Making an Agarose Gel

An agarose gel is prepared by combining agarose powder and a buffer solution. Flask for boiling Agarose

Electrophoresis Equipment Power supply Cover Gel tank Electrical leads  Casting tray Gel combs

Preparing the Casting Tray Place the casting tray on a level surface. The gel combs should NOT be touching the surface of the casting tray.

Pouring the gel Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles. the gel combs should be submerged in the melted agarose solution.

When cooled, the agarose polymerizes, forming a flexible gel When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape.

Place the gel in the electrophoresis chamber.

DNA buffer   wells    Anode (positive) Cathode (negative) Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.

Sample Preparation Mix the samples of plasmid with the 6X sample loading buffer. This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells. 6X Loading Buffer:   Bromophenol Blue (for color)  Glycerol (for weight)

Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.

Running the Gel Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.

Cathode (-)  wells  Bromophenol Blue Gel Anode (+) DNA (-)  After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.

DNA Ladder Standard -  250  5,000  2,500  1,000  15,000 bp  10,000  7,500 DNA migration Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule bromophenol blue + Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.

Staining the Gel • Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. • Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run. ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times.

Staining the Gel • Place the gel in the staining tray containing warm diluted stain. • Allow the gel to stain for 25-30 minutes.

Ethidium Bromide requires an ultraviolet light source to visualize

Open circular linear supercoiled