This is a scientific report. You need to do only introduction, method and reference section.
Practical 1: DNA manipulation
Background
In this session, you will perform a plasmid mini-prep to isolate pBlueScript KS II (+) (pBKS II) (Fig 2) from E. coli. You will then perform a double restriction enzyme digestion on pProExTM with the enzymes BamHI and HindIII to release the FtsZ insert. The pBKS II DNA that you isolate will need to be prepared to accept the insert by restriction enzyme digestion with the same restriction enzymes. Next week, we will ligate the Pc-ftsZ gene into pBKS II.
Exercise 1A: Small-scale plasmid isolation (‘Miniprep')
Objective
To isolate the pBlue Script KS II (+) plasmid, from an overnight culture of E.coli.
Introduction
Plasmids are extra-chromosomal genetic elements found in a variety of bacterial species. They are double-stranded, closed circular DNA molecules that range in size from 1 kilo base (kbp) to greater than 200 kbp.
Plasmids often contain genes encoding enzymes that are advantageous to the bacterial host (i.e. resistance to antibiotics, degradation of organic complexes).
One common reason to isolate plasmid DNA from a host cell is to use it as a vector or cloning vehicle. By a transformation procedure, a particular DNA segment of interest, or random piece of DNA, is inserted (joined/ligated) into a plasmid and the recombinant molecule is then placed in a cell (in this case E. coli) in which replication of the plasmid and DNA of interest can occur.
All methods of plasmid DNA isolation involve these basic steps:
- culture of host cells containing plasmid,
- harvesting and lysis of the host cells, and
- Protein and chromosomal DNA precipitation
- Purification of the plasmid DNA.
These exploit in one way or another, the two major differences between chromosomal and plasmid DNA:
1. Chromosomal DNA is much longer than the DNA of plasmids commonly used as vectors.
2. The large DNA molecules (chromosomal DNA) and proteins form precipitates because they bind to each other in a large aggregate but the plasmids don't precipitate because they renature correctly and don't become part of the large multi-molecule aggregates. Thus plasmid DNA remains in solution while proteins and other DNA molecules precipitate. It is subsequently precipitated using ethanol or isopropanol.
Method
Refer to the demonstration flow diagram for an overview of the method. Students should perform a plasmid preparation in pairs.
1. Inoculate 3 mL of LB (Luria Broth) + ampicillin with a single colony of bacteria containing pBlueScript II KS+ (AmpR). Grow at 37°C overnight (This step has been done for you).
2. Transfer 1 mL of the bacterial culture into a microcentrifuge tube and centrifuge for 1 min at max. speed (Caution. Risk group 1 Microorganism. Wear gloves. Risk = Low). Do not discard remaining culture. (Caution. Centrifuge must be weight balanced. Place samples evenly around the rotor. Use a tube with 1.5mL of water in to balance the samples if the number is uneven. Use rotor lid. Do not operate without supervision).
3. Pipette off the supernatant and discard it in the liquid waste container. Do not disturb the pellet but try to leave the pellet as dry as possible. Repeat steps 2 and 3, i.e. double the amount of pelleted cells if your pellet appears small (check with your demonstrator).
Isolate plasmid DNA using the following procedure:
NOTE: Timing must be exact. Wait for the demonstrator to coordinate the procedure. If you take too long your DNA will permanently denature.
4. Resuspend your cells in 100 µL of GTE Buffer. Pipette the cells up and down. Make sure there are no clumps of cells and the solution is homogeneous. Vortex the solution if necessary.
5. Add 200 µL of Lysis Buffer (at room temp). (Warning. Contains sodium hydroxide and SDS. Corrosive. Irritant. Wear gloves, safety glasses and mop up spills. Risk = Low). Rapidly mix by inverting the tube 5 times, and then incubate on ice for 5 min.
6. Add 150 µL of ice-cold Neutralisation Buffer. Mix by inverting 5 or 6 times, then incubate on ice for 5 min.
7. Centrifuge at top speed for 10 min and transfer 400 µL of supernatant to a new tube. (Label as pBKSII plasmid Tube #1)
Note: You should not carry over any white precipitate from this step. If you notice that you have, centrifuge the sample a second time for 5 minutes and remove the clear supernatant.
8. Precipitate DNA by adding 2.5 times (step #7) volumes (1000µL) of 100% ethanol (Caution. Flammable. Irritant. Mop-up. Risk = Low). Mix by gentle inversion 5 or 6 times
9. Centrifuge at maximum speed for 10 min.
Tip: place the tube in the microfuge with the hinge facing to the outer edge. When you remove the tube your DNA pellet should be near the base of the tube one the same side as the hinge.
10. Carefully remove the supernatant with a pipette, and discard the liquid into the waste container. Do not dislodge the DNA, which should be visible as a clear to whitish pellet.
11. As a wash, add 1 mL (1000 µl) of 70% ethanol (Caution. Flammable. Mop-up. Risk = Low) to the pellet, invert the tube several times, then centrifuge at top speed for 2 min.
12. Dispose off supernatant.
13. As a final wash, add 500µl of 70% ethanol (Caution. Flammable. Mop-up. Risk = Low) to the pellet, invert the tube several times, then centrifuge at top speed for 2 min.
14. Carefully pipette off the supernatant (again, don't dislodge pellet). Ensuring as much ethanol as possible has been removed. Invert the tube on a piece of paper towel and allow the residual ethanol to evaporate (this may take 10-15 min).
Warning: Dry well. Do not leave residual ethanol in the tube. Ethanol will inhibit further enzymatic reactions.
15. Resuspend the pellet in 40 µL of 10 mM Tris-HCl (pH 8.0) with RNase A (Elution Buffer EB). Mix by flicking the tube. Incubate for 10 min at 37°C to assist the process. Label as Tube #1
16. Aliquot 5 µL of the plasmid sample into a sterile microfuge tube, and clearly label this tube "5 µL pBKS II - Southern blot" with your group name and date. Label as Tube #2 Place in your group's freezer bag which will be stored at -20°C until next week. Retain the remaining 35 µL plasmid sample for the restriction enzyme digestion.
1. Why is ampicillin included in the growth medium?
2. Why is RNase A used in the procedure?
Exercise 1B: Restriction endonuclease digestion of the donor and recipient plasmid
Objective
*To digest the donar plasmid pProEX plasmid with BamHI and HindIII and excise the Pc- FtsZ gene
*To prepare pBKSII to receive the Pc-ftsZ gene by digesting it with BamHI and HindIII
Introduction
Restriction endonucleases (REs) are bacterial enzymes that cleave double-stranded DNA. Type 2 REs described for use here require highly specific sites for DNA cleavage and thus are extremely useful and selective tools.
REs are isolated from a variety of bacterial strains (approximately 2,000 are known) and are present in bacteria, presumably to destroy DNA from foreign sources (e.g. infecting bacteriophage) by cleaving the foreign DNA at specific recognition sites. Being enzymes, each has specific requirements for optimal cleavage activity (i.e. temperature, ionic strength of buffers).
Unlike pBKSII, pProEXTM is a low-copy plasmid. These plasmids need to be prepared at a larger scale than you can perform in this practical. The pProEXTM DNA has been prepared for you.
1. Determine the predicted fragment sizes (in bp) of a digestion of the pBluescript II KS (+) plasmid (Fig 3) using BamHI and EcoRI.
Attachment:- Practical Report.zip