Skip to main content

Chromosomal DNA Purification

Extraction of chromosomal DNA from eukaryotic cells is the common procedure in molecular biology experiments and forensic analysis. Chromosomes of eukaryotic cells are located within the nucleus of the cell. Each eukaryotic chromosome consists of a single DNA molecule wrapped around the histone proteins. In order to get the chromosomal DNA out of the cell following steps are used. 
1) Cells are treated with detergents such SDS (Sodium Dodecyl Sulfate), Triton-X-100 or Tween in order to break open the cell. These detergents will break the plasmamembranes and nuclear membrane of the cell and cytoplasmic material & proteins are digested. (The membranes are dissolved using detergents that solubilize the phospholipids that make up these membranes). 

2) Once the membranes are dissolved, with the help of centrifuge, the cell lysate is formed. This cell lysate contains the cytoplasmic material, organelles and nuclear material and chromosomes.

3) The proteins are then treated with the enzyme called Proteases such as Protease K which will digest these proteins. 

4) After the protease treatment, the DNA will be present in Aqueous Solution.

5)Then the cell lysate is treated with the mixture of phenol and chloroform which are organic solvents. This mixture will remove the proteins and other cell debris. They leave the DNA in the aqueous interphase between phenol and chloroform. 

6) The above step is repeated to further purify the DNA. (When absolute 100% ethanol is layered on top of the aqueous DNA solution, the interphase develops between two liquids due to their difference in densities). 

7) DNA in the aqueous solution (interpase) is then spooled using the glass rod or pipette. This pipetted solution is then transferred to fresh and clean tube. where the DNA can be dried and stored or can be diluted with aqueous solution and stored. 

8) This purified DNA is then used for further experiments such PCR, gene analysis, cloning or molecular biology experiments. 


Image result for dna

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

Protocol: Chromosomal DNA Isolation from Bacteria

1.       Spin down 50-100 ml well-grown bacteria, 3600 rpm,15min. 2.     Resuspend bacteria with 20 ml Buffer S, immediately add 100 µl ProteinaseK (10 mg/ml). Vortex to make sure no chunks. 3.       Add 2 ml of 20% SDS, mix gently by inverting. 4.       Incubate the mixture at 65 o C for 1 hr with inverting every 15 min. 5.     Add 10 ml of phenol and 10 ml of chloroform, mix thoroughly by inverting for 5 min, spin at 3600 rpm for 20 min. 6.       Transfer supernatant to a new tube, add 0.6 volume of isopropanol. Mix gently by inverting. You will see cotton-like genomic DNA. 7.       Hook out the cotton-like DNA to a 1.5 ml tube; wash with cold 70% ethanol. 8.      Dry DNA at RT, dissolve DNA in 500 µl H 2 O (50 o C or 4 o C overnight). 9.      Add 5 µl DNase-free RNase A (20 mg/ml stock) to the DNA. Incubate at 37 o C for 30 min. 10.   Add 500 µl phenol, mix, spin at 3600 rpm for 20 min. Repeat phenol extraction once if necessary. 11.   Tr
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