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  Section: Plant Lab Protocols
 
 
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Methodology for Nucleic Acids

 
     
 
Isolation of plasmids
 
Plasmids are extrachromosal, self-replicating double-standard, circular DNA molecules found in most prokaryotes. These molecules carry genetic information for a variety of special functions such as resistance to antibiotics, nitrogen fixation, ability to utilize novel substrates etc. The plasmids can be transferred from one cell to another and therefore function as vectors or carriers in genetic engineering techniques.
 
A number of plasmids used in genetic engineering have a relaxed mode of replication. This means that the plasmid replicates independently of chromosomal control, accumulates up to one-third of the cellular DNA content when cell protein synthesis is inhibited by a drug. This milligram quantities of plasmid DNA may be isolated from a single liter of cells.
 
A variety of procedures are available for the isolation of plasmid DNA. The choice of the method depends upon the source material, the purity of the DNA required, the number of samples etc.
 
For cloning work the plasmid DNA is required in substantially pure form and involves elaborate procedure. On the other hand, a large number of samples can be examined by a 'rapid method' for qualitative information. A procedure for culturing and harvesting of bacterial cells, lysing of cells and extraction of plasmid, is given below.
 
 



Principle

The bacterial cells are grown to stationary phase, harvested and gently lysed by weakening the cell walls with lysozyme treatment followed by use of the detergent SDS. As a result the cells release their DNA in high molecular weight form which is removed by high speed centrifugation leaving the plasmid DNA in the cleared lysate. This fraction is deproteinized and nucleic acids are then precipitated by ethanol Purification of the plasmid is performed by equilibrium density centrifugation in cesium chloride.
 
 



Materials

Bacterial Strain carrying the Plasmid (e.g., E. coli JA 221 carrying pBR 328)
LB Broth
   Yeast extract                      5g
   NaCl                                    10g
   Tryptone                             10g
   Water                                  1L
TE + Sucrose (pH 8.0)
   0.05MTris                           0.61g
   25% (w/v) sucrose            25g
   Water                                  100mL
Lysozyme Solution: 5mg/mL in 0.25M Tris-HCl (pH 8.0)
Phenol-Chloroform Mixture: 1:1 (v/v)
Saline Sodium Citrate (SSC) solution:
   0.15 M NaCl                      0.88g
   0.015M sodium citrate    0.44g
   Water                                 100mL
   Dilute it ten times to get 0.1 SSC.

Ethidium Bromide 5mg/mL in 0.1 SSC solution
0.25M EDTA solution
TES Buffer (pH 8.0)
   30mM Tris                          0.36g
   5mM EDTANa2+                0.19g
   50mMNaCl                        0.28g
   Water                                  100mL
High-speed Refrigerated Centrifuge
Ultracentrifuge with suitable Rotors
Polycarbonate Ultracentrifuge Tubes
UV Lamp (longwave length)
Pasteur Pipettes
 
 
Procedure
A. Harvesting ceils
1.
Grow the bacterial strain in 250mL LB broth + antibiotic (ampicillin) at 37°C with shaking (vigorous aeration) to stationary phase.
2.
Harvest the cells by centrifugation at 5,000rpm in a refrigerated centrifuge for 10 min at 4°C.
3.
Wash the cells by resuspending in the TES buffer and centrifuging at 6,000rpm for 10 min. Repeat the washing step.
4.
Resuspend the cells in a small volume of TE + Sucrose buffer. (The cells can be stored frozen at this stage, if necessary.)   Make up the volume of suspension to 3.75mL by adding TE + Sucrose buffer.
 
B. Lysing Cells and DNA Isolation
5.
Transfer the cell suspension to a pre-cooled 100mL flask.
6.
Add 0.75mL of lysozyme solution followed by 1.25mL of 0.25M EDTA (pH 8.0) solution and shake the contents on ice for 10 min.
7.
Add 0.75mL of 20% SDS (final concentration 2%) and ensure uniform mixing.
8.
Incubate without shaking at 37°C in a water bath until the suspension clears (cell lysis; 10-60 min). Cool on ice.
9.
Centrifuge the lysate in thick-walled polycarbonate tubes in an ultracentrifuge at 40,000rpm for 1h at 20°C. (Use 0.25 Tris pH 8.0 for balancing the centrifuge tubes, if necessary.) This will clear the lysate and the supernatant will contain most of the plasmids with RNA and proteins as contaminants. High molecular weight chromosomal DNA
is removed in the pellet.

10.
Carefully decant the supernatant into a measuring cylinder, note its volume and transfer into a 100mL flask. Add 0.1 volume supernatant 2.0 M Tris base (pH unadjusted).
11.
Add an equal volume of phenol: chloroform. Shake thoroughly at room temperature for 4 min.
12.
Centrifuge the emulsion in a bench centrifuge at 5,000rpm for 10 min to separate the aqueous and organic phases.
13.
Transfer the upper aqueous phase to fresh flask using a Pasteur pipette taking care not to disturb the protein precipitate at the interface. Repeat steps 11 and 12.
14.
Carefully remove the aqueous phase and not its volume. Add 0.25 times the volume of 4.5M potassium acetate to give a 0.9M solution to ensure quantitative precipitation of DNA.
15.
Add two volumes of chilled ethanol and place in freezer for 60min to allow complete precipitation of DNA.
16.
Centrifuge the contents at 10,000rpm for 10 min at 0°C to pellet the DNA. Decant the supernatant and drain off any liquid by inverting the tubes on paper towels.  Dry gently in a vacuum desiccator or using a stream of nitrogen gas.
17.
Dissolve the precipitate in 0.4mL 0.1 SSC and withdraw 2QaL for testing by electrophoresis; then make up the remaining solution to 3.6mL with 0.1 SSC.
 
 
Purification by Cesium Chloride Centrifugation
18.
Dissolve 3.9g CsCl in the preparation completely. Then add 0.4mL of ethidium bromide.
19.
Load the sample into ultracentrifuge tubes to within a few mm of the top and balance the tubes in pairs.
20.
Centrifuge at 140,000g for 40h at 20°C in a swing-out rotor.
21.
After centrifugation view the tubes under long-wave UV light. The DNA-ethidium bromide complex fluoresces and two defined bands could be seen near the middle of the tube. The more intense lower band consists of supercoiled, circular plasmids and the top band consists of linear plasmids and fragments of nuclear DNA.
22.
The plasmid band can be recovered by a number of ways. First draw-off the upper part of the gradient and the top DNA band using a Pasteur pipette. Then suck the plasmid band into a sterile syringe fitted with a wide-bore needle carefully.   Alternatively, a long needle fitted to a syringe is carefully lowered to the plasmid band and carefully drawn
into the syringe.

23.
Remove the ethidium bromide from the plasmic fraction by extracting thrice with two volumes each of isopropyl alcohol. Cesium chloride and ethidium bromide are removed by dialysis for 16h against several changes of 0.1 x SSC or any other suitable buffer for future analysis.
24.
Following dialysis, transfer the plasmid solution to sterile tubes. Measure the absorbance at 260 and 280nm. The A260 should be nearly two-fold of A280 for a good preparation. Calculate the concentration of plasmid DNA using the relationship A260 of 1.0 = 5Qag/mL of DNA. The preparation can be stored frozen for several weeks.   If a more concentrated preparation is required, concentrate by precipitation with ethanol (steps 14-16).
 
 
Notes
1.
Exercise enough care while handling the live bacterial cells in order to avoid any contamination and dispose properly.
2.
After treatment with SDS the bacterial suspension should become highly viscous and gel-like indicating successful lysis. Treat the suspension as gently as possible to avoid damaging unwanted high MW DNA released from the cells.
3.
Dissolve the DNA precipitate, which may be even invisible in the centrifuge tubes, very gently to avoid shearing of DNA molecules.
4.
For a good resolution in CsCl gradient, ultracentrifugation should preferably be done in rotors that take short, wide tubes.
5.
Ethidium bromide and ultra-violet light are harmful; wear gloves and safety glasses respectively while using them.
6.
The dialysis tubing used in the procedure should be pre-treated by boiling in 10mM EDTA for 15 min followed by two 15 min treatments in boiling distilled water.
7.
At appropriate stages aliquots may be withdrawn and analyzed on agarose minigels to follow the course of purification of plasmid DNA.
8.
The linearized plasmid DNA molecules after calating with ethidium bromide band slightly above the intact molecules in the gradient.
 
 
References
1.  Clewell, D B and Helinski, D R (1971) Biochem 9 4428.
2.  Maniatis, T, Fritsch, E F and Sambrook, J (1982) Molecular Cloning - A Laboratory Manual Cold Spring Harbour Laboratory New York.
 
     
 
 
     




     
 
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