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  Section: Biotechnology Methods » Molecular Biology
 
 
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Transformation

 
     
 
Content
Molecular Biology
  The Central Dogma
  Protein Synthesis in Cell Free Systems
  Chromosomes
  Polytene Chromosomes of Dipterans
  Salivary Gland Preparation (Squash Technique)
  Extraction of Chromatin
  Chromatin Electrophoresis
  Extraction and Electrophoresis of Histones
  Karyotype Analysis
  In Situ Hybridization
  Culturing Peripheral Blood Lymphocytes
  Microslide Preparation of Metaphases for In-Situ Hybridization
  Staining Chromosomes (G-Banding)
  Nucleic Acids
  Extraction of DNA from Bovine Spleen
  Purification of DNA
  Characterization of DNA
  DNA-Dische Diphenylamine Determination
  Melting Point Determination
  CsCl-Density Separation of DNA
  Phenol Extraction of rRNA (Rat liver)
  Spectrophotometric Analysis of rRNA
  Determination of Amount of RNA by the Orcinol Method
  Sucrose Density Fractionation
  Nucleotide Composition of RNA
  Isolation of Genomic DNA—DNA Extraction Procedure
  Isolation of Genomic DNA from Bacterial Cells
  Preparation of Genomic DNA from Bacteria
  Extraction of Genomic DNA from Plant Source
  Extraction of DNA from Goat Liver
  Isolation of Cotton Genomic DNA from Leaf Tissue
  Arabidopsis Thaliana DNA Isolation
  Plant DNA Extraction
  Phenol/Chloroform Extraction of DNA
  Ethanol Precipitation of DNA
  Isolation of Mitochondrial DNA
  Isolation of Chloroplast DNA
  DNA Extraction of Rhizobium (CsCl Method)
  Isolation of Plasmids
  RNA Isolation
  Preparation of Vanadyl-Ribonucleoside Complexes that Inhibit Ribonuclease Activity
  RNA Extraction Method for Cotton
  Isolation of RNA from Bacteroids
  Isolation of RNA from Free-Living Rhizobia
  Estimation of DNA purity and Quantification
  Fungal DNA Isolation
  Methylene Blue DNA Staining
  Transformation
  Blotting Techniques—Southern, Northern, Western Blotting
  Preparing the Probe
  Southern Blotting (First Method)
  Southern Blotting (Second Method)
  Western Blotting
  Western Blot Analysis of Epitoped-tagged Proteins using the Chemifluorescent Detection Method for Alkaline Phosphatase-conjugated Antibodies
  Southern Blot
  Southern Analysis of Mouse Toe/Tail DNA
  Northern Blotting
  Restriction Digestion Methods—Restriction Enzyme Digests
  Restriction Digestion of Plasmid, Cosmid, and Phage DNAs
  Manual Method of Restriction Digestion of Human DNA
  Preparation of High-Molecular-Weight Human DNA Restriction Fragments in Agarose Plugs
  Restriction Enzyme Digestion of DNA
  Electroelution of DNA Fragments from Agarose into Dialysis Tubing
  Isolation of Restriction Fragments from Agarose Gels by Collection onto DEAE Cellulose
  Ligation of Insert DNA to Vector DNA
  PCR Methods (Polymerase Chain Reaction)
  Polymerase Chain Reaction
  DNA Amplification by the PCR Method

Introduction
Introduction of recombinant plasmid into cells is achieved by the transformation of competent cells. Competent cells are prepared by treating the cell with a divalent cation like calcium chloride. Once the cells are made competent, the plasmid DNA is mixed with the cells. The competent cells are then subjected to heat shock, which allows the DNA to enter the cells. The cells are then plated onto a medium containing antibiotics to allow identification of recombinants.


Competent Cells Preparation

The competent cells were prepared by the following methods:
  • Calcium chloride method
  • Polyethylene glycol 80(x) method
  • Calcium chloride method
  • Material calcium chloride (50 mM), E. coli cells, ice, 10% sterile glycerol,
    bullets, tips, LB
  • Agar plate, LB broth, etc.

Procedure

E. coli strain cells were made competent for transformation by treating them with calcium chloride, as described by Sambrook et al (1989). Bacteria in glycerol frozen stock were streaked on a LB agar plate using a sterile platinum loop, grown at 37°C for 16–20 hours. A single isolated colony from the plate was transferred into 25 mL of LB broth and grown for 16 hours at 37°C with shaking until the culture reached an A600 nm of 0.4.

The culture was chilled on ice and the cells were harvested by centrifuging at 4000 rpm for 10 minutes, at 4°C in a refrigerated centrifuge. The cell pellet was resuspended in 25 mL ice-cold sterile 50 mM calcium chloride and kept on ice for 20 minutes. Cells were recovered by centrifugation at 4°C as described above. Finally, the pellet was gently resuspended in 2.5 mL of ice-cold 50 mM calcium chloride containing 10% sterile glycerol, aliquoted (200 mL each) in °C for further use.


Transformation of Competent Cells
  1. Mix 10 mL of ligation mix (or any other plasmid that is to be transformed) with 100 mL of competent cells and incubate on ice for 1 hour.
  2. Apply a heat shock at 42°C for 2 minutes.
  3. Chill the tube to 0°C on ice immediately.
  4. Add 800 mL of sac medium and incubate at 37°C for 60 minutes with slow shaking.
  5. Prepare LB-agar plates by adding 2 mL ampicillin (50 mg/mL), 10 mL IPTG, and 10 mL X.
  6. Stock per mL of melted LB agar.
  7. Spread 100 mL of transformed cells onto the plates and incubate at 37°C overnight.

Screening of Recombinants
  1. Selection of recombinants is based on the color of the colony.
  2. For a pUC vector, since the site used for insertion of foreign DNA is located within the lacZ gene, insertion of foreign DNA is monitored by the loss of 13-galactosidase activity upon transformation. Cells with the intact lacZ gene produce functional 13-galactosidase, which converts the colorless substrates X-gal to blue chromophor in presence of an inducer IPTG and therefore produce blue colonies. Transformed cells with recombinant plasmid do not demonstrate 13-galactosidase activity, and therefore, cannot act on X-gal resulting in the production of white colonies.
  3. Select white colonies as clones. Storage of Clones
  4. Transfer the white colonies one by one onto a fresh LB agar plate containing ampicillin, IPTG, and X-Gal.
  5. Incubate the plate at 37°C overnight. This is the master plate of the clones.
  6. Inoculate the clones from the master plate to 1 mL of LB containing 100 mL of ampicillin individually.
  7. Incubate the tubes at 37°C overnight with constant shaking.
  8. Add 150 mL of 100% glycerol to the wells of the microtiter plate.
  9. Add 850 mL of the overnight grown culture to glycerol in a microtiter plate. Use 1 well per clone. Mix well.
  10. Freeze the plate, cover, and store at –70°C.
Notes
  1. The competent cells prepared may be stored frozen at –70°C without loss of activity for long time. Store in small aliquots and take out a fresh tube and use for transformation whenever required.
  2. The procedure for plating is given for plasmids with lacZ as an insertional inactivation marker and ampicillin as a selection marker. For plasmids with other markers, prepare the plating media accordingly.
Observations
Count white colonies as recombinant transformants and test for insert. Calculate the transformation efficiency in terms of the number of colony-forming units (CFU) per microgram of transforming DNA as follows:
CFU on Plate
CFU/yg = _____________× 101 × dilution factor ng plasmid DNA used in transformation


 
     
 
 
     




     
 
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