Skip to main content

Plasmid DNA Isolation from Bacteria


In bacteria, the Plasmid is present in the cytoplasm. To get the plasmid DNA out of the bacteria, its cell membranes must be disrupted in order to get the Plasmid DNA in the extraction buffer. There are three main solutions involves in the procedure. 

Solution I: It contains Glucose, Tris and EDTA.. Glucose provides the Osmotic shock which leads to the disruption of the cell membrane. Tris  is buffering agent and maintains constant pH 8. EDTA is used to protect the Plasmid from endogenous nucleases by chelating the magnesium ions. 

Solution II: it contains NaOH and SDS. This is an Alkaline solution. It is used to disrupt the cell and NaOH also denatures the DNA into single strands. 

Solution III: it contains the acetic acid and potassium acetate. Acetic acid is used to neutralize the pH. and Potassium acetate is used to precipitate the chromosomal DNA, proteins and other cellular debris. 

Phenol/Chloroform: It is used to denature and separate proteins from Plasmid. Chloroform stabilizes the boundary between an aqueous phase and pure phenol layer. Denatured proteins form a layer at the interface between aqueous and organic phases, which are removed by the centrifugation. 

(Once the plasmid is released it must be precipitated with alcohol such as cold ethanol or isopropanol). 

Procedure:
  • Take 2mL of overnight culture and centrifuge for 5 minutes. Discard the supernatant carefully. Do not disturb the pellet.
  • Add 100µL of Solution I to the pellet and re suspend the cells using pipette.
  • Incubate the mixture for 5 minutes at room temperature.
  • Add 200µL of Solution II to the mixture and mix by inverting the tubes for 5 minutes.
  • Incubate the mixture for 5-10 minutes at room temperature.
  • Add 500µL of ice cold Solution III to the mixture and mix by inverting the tube.
  • Incubate on ice for 10 minutes.
  • Centrifuge the tubes at 10,000 rpm for 5 minutes.
  • Transfer the supernatant into a fresh tube.
  • Add 400µL of phenol-chloroform mixture to the contents and mix well by inverting the tube for 3-4 times and incubate at room temperature for 5 minutes.
  • Centrifuge the tubes at 10000 rpm for 5 minutes.
  • Collect the viscous supernatant using cut tips and and transfer it to a fresh tube.
  • Add 800µL of isopropanol and mix gently by inversion. Incubate it for 30 minutes at room temperature.
  • Centrifuge at 10,000 rpm for 10 minutes.
  • Discard the supernatant.. Do not disturb the pellet.
  • Keep the tubes open for air drying for 5-10 minutes.
  • After air drying, add 100µL of TE buffer or distilled water and resuspend. (The contaminated salt in the DNA can be removed with 70% ethanol washing). 
Plasmid DNA Isolation
Image Source: srmuniv

Labels:

Comments

Post a Comment

Popular posts from this blog

Statistics in Flow Cytometry Data and "MFI" values

                                 The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity..                Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values.  (Keeping in mind that, consideration of statistics in flow cyomtery also depends on the type of Application you are interest
Post a Comment
....CONTINUE READING

Gene Cloning Technique

Gene Cloning Technique in Molecular Biology Field Involves the following steps: 1.a) Isolation of Genetic Material and Gene Sequence of Interest.  First, cells are lysed using detergent or lysozyme enzymes which disrupts the plasma membrane and release the genetic material along with the macro molecules such proteins and RNA molecules. Cell contents are then treated with protease to disrupt the proteins and RNase to destroy the RNA. Cell debris are then pelleted using centrifuge and supernatant containing DNA is transferred to a fresh and clean tube. A proper amount of Ethanol is added to this supernatant and precipitated using centrifuge. Supernatant is discarded and the pellet which has DNA is suspended using a proper suitable buffer.  Primers are designed for the specific gene sequence of interest which will be used for the cloning procedure.  Gene sequence is then amplified using PCR (Polymerase Chain Reaction) which will yield in many copies of the gene sequence.
Post a Comment
....CONTINUE READING

Nanodrop

Thermofisher, Nanodrop 1000    Nanodrop is a spectrophotometer which is designed to measure the concentration of nucleic acids at 260nm and Proteins at 280nm present in a very small minute volume. It is also capable of measuring the concentrations of dsDNA, RNA, ssRNA and purified protein. Required sample size is usually 1µL. Nanodrop instruments are usually full spectrum spectrophotometer, i.e., Ultra violet and visible range wavelengths (250nm to 750nm).  Nanodrop is commonly used for the quantification of the DNA concentration and RNA concentration in a given sample. However there are many other applications which can be performed on this instrument.  It works on the principle of Spectrophotometer or Sample Retention System....  Principle of Nanodrop: "The Nanodrop works on the principle of sample retention system.. When the small volume of sample is introduced, the arm and pedestal will work together and make the sample in the form of column and forms approxi
Post a Comment
....CONTINUE READING