1. Plasmid preparation and Restriction digestion

2. Exp 1 Plasmid mini-preparation from E. coli

3. 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.

5. 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

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

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

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

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

10. 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

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

12. 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.

13. Restriction Enzymes digestion and agarose gel electrophoresis

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

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

16. 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

17. 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

18. Agrose Gel Electrophoresis

19. 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.

20. + - 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

21. + - 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

22. 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

23. 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.

24. Making an Agarose Gel

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

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

27. 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.

28. 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.

29. 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.

30. Place the gel in the electrophoresis chamber.

31. 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.

32. 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)

33. 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.

34. 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.

35. 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.

36. 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.

37. 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.

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

39. Ethidium Bromide requires an ultraviolet light source to visualize

40. Open circular
linear
supercoiled