Genomic DNA Isolation from Bacteria

Protocol
1) Take 1.5ml of the bacterial culture, centrifuge at 14,000 rpm for 5mins.
2) Decant the supernatant completely & make a single cell suspension.
3) Add 500ul of 10mM Tris, 10mM EDTA, 100mM NaCl, 2% SDS, 20ug/ml of proteniase K.
4) Mix well and incubate at 560C for 30mins.
5) Add 500ul isopropanol, mix well & centrifuge at 14,000rmp for 10mins.
6) Decant the supernatant & add 200ul of 80% ethanol, centrifuge at 14,000rpm for 10mins at 40C.
7) Repeat the above step.
8) Air dry the pellet.
9) Dissolve in 200ul of sterile distil water.
10) Run the samples on 0.8% agarose gel.

Note: Phenol Chloroform purification can be performed to get pure DNA preparation.

Bacterial Cell Induction using IPTG and Extraction of Protein

Protocol

A) Induction of Bacterial culture.

1) Take 50ml of LB broth and add the required antibiotic in the suitable concentration and 2ml of the culture.
2) Incubate at 370C at 220 rmp, till the OD at 600nm reaches 0.8-1.
3) Once the OD is reached add 0.2mM IPTG to the above culture.
5) Incubate at 370C at 220 rmp for 1-1:30 hrs.
4) Store at 40C till the extraction process.

B) Extraction of Protein from the Induced Culture.

1) Transfer 50ml of induced and uninduced (as negative control) in 50ml tube.
2) Centrifuge at 14,000rpm for 1min or 5,000rpm for 5mins at 40C.
3) Completely decant the supernatant and dislodged the pellet to make a single cell suspension.
4) Take 10ml of Binding Buffer and to that add 10ul of Triton-X100 + 10ul of cocktail of Protease Inhibitor.
5) Add 2ul of the above mix to the single cell suspension.
6) Vortex properly for about 10-20secs.
7) Transfer the above solution to a eppendrof.
8) Dis rupture the cells using sonicator at 35 amplitude for 1min with pulse on for 1sec and pulse off for 1sec.
9) After sonication, incubate the on ice for 15mins.
10) Centrifuge for 30mins at 12,000rpm, 40C.
11) Transfer the supernatant on another eppendroff, and store the pellet at -800.
12) Load the supernatant on the column bases on the property of the protein need to be isolated and collect the flow through.
13) Add 10 volumes of Binding buffer based on the column height.
14) Wash with wash buffer and collect the wash.
15) Pass different concentrations of Imidazole Elution Buffer and collect elution.
16) Run all the flow through and elution on SDS-PAGE.

C) Preparation of the Column

1) Take a 5ml syringe and seal one end of the using glass wool.
2) Add some water to the column and over that slowly from the sides add HisPur Cobalt Resin.
3) Fill the column with resin up to 1ml marks in the syringe.
4) Wash the column with 3 ml of water.
5) Equilibrate the column with 3 volumes of  Binding buffer based on the amount of resin added.
6) The column is ready to be used.
7) Once all the samples are run, we should pass 100mM EDTA through the column. This process is called as stripping of the column and the column is ready for the next use and after a repeated use for a long time. we can again charge the column using cobalt.
8) Seal both the ends of the column using parafilm. and store at 40C till further used.

NOTE: At any point during the experiment the column should not be dry.

Solutions

1) Binding Buffer (pH 7.8)
 20mM Sodium Phosphate
50mM NaCl

2) Wash Buffer (pH 6)
20mM Sodium Phosphate
50mM NaCl

3) Imidazole Elution Buffer (pH 6)
20mM Sodium Phosphate
50mM NaCl
100mM, 200mM, 300mM, 400mM and 500mM Imidazole

4) 20mM Sodium Phosphate
pH            Volume of Na2HPO4(ml)       Volume of NaH2PO4(ml) 
7.8            89.6                                                         10.4
6.0            12.0                                                         88.0
Dilute the 1M stock solution to 1liters with distil water.

Polytene Chromosome

Drosophila is an excellent genetic research organism because fruit flies:
• have a short generation time (important for research spanning a number of generations)
• are small and easy to keep in a laboratory
• produce reasonably good numbers of offspring
• have a number of easy to see inheritable characteristics
• have a chromosome number of 8 (4 pairs of chromosomes)

Insects typically have multiple life cycle stages, including egg, larva, pupa and adult. The fruit fly is typical of those insects whose larval stage is the assimilative stage. Larvae may pass through several stages before they pupate. After pupating for the requisite time, adults hatch out and find mates. Adults generally are short-lived.

Many larval and some adult tissues of insects in the family Diptera are characterized by nuclei with giant chromosomes. These chromosomes develop by multiple replications of the chromosomes within each cell during development. Each nucleus will contain hundreds of copies of each chromosome. Cells are considered polyploid if they have more than two copies of each chromosome. If the chromosomes align perfectly forming large cables of chromosomes they are polytene.

In Drosophila melanogaster, chromosomes of the larval salivary gland contain about 1024 copies of the DNA, or ten doublings from the normal 2n condition, of each of the three chromosomes. Each gene is exactly aligned with its homologs on the other 1023 copies. The pattern of condensed regions (heterochromatin), and transcribed regions (euchromatin) gives a series of about 5000 light and dark bands when the chromosomes are stained with orcein. The banding patterns of the chromosomes show significant phylogenetic and ontogenetic stability. Genetic maps relate these bands to their functions. In general, the DNA in each band codes for a single function, although there are exceptions to this observation. Drosophila has given us substantial insight into DNA function and gene organization 

Polytene chromosomes are giant chromosomes common to many dipteran (two-winged) flies. They begin as normal chromosomes, but through repeated rounds of DNA replication without any cell division (called endoreplication), they become large, banded chromosomes. For unknown reasons, the centromeric regions of the chromosomes do not endoreplicate very well. As a result, the centromeres of all the chromosomes bundle together in a mass called the chromocenter.

Polytene chromosomes are usually found in the larvae, where it is believed these many-replicated chromosomes allow for much faster larval growth than if the cells remained diploid. Simply because each cell now has many copies of each gene, it can transcribe at a much higher rate than with only two copies in diploid cells. 

The polytene chromosomes at the right are from the salivary glands of the fruit fly Drosophila melanogaster. the bands on each chromosome are like a road map, unique to each chromosome and well defined enough to allow high resolution mapping of each chromosome.

Procedure

1. Prepare a clean slide with 2-3 drops of PBS (Phosphate Buffer Saline) and put the slide (without a cover slip) on a stereoscopic (dissecting) microscope.
2. Select a large Drosophila melanogaster larva and place it on the slide.
3. While looking through the microscope use probes or forceps to grasp the larva by its
   midsection just behind its jaws.
4. Gently stretch the larva by pulling on it until its head separates from the rest of its body.
5. Look for the salivary glands in the head section. The glands are very small, fairly
   transparent, usually paired and have dark fat particles attached.

6. When you have located the salivary glands, separate them from the rest of the fruit fly
tissues. Once you are certain that you have successfully done this, you may dispose of
the rest of the larva appropriately. Keep the salivary glands moist with PBS at all
times. Do not let it dry.

7. Add 2-3 drops of aceto-orcein stain to your Drosophila salivary glands and keep it for 5-10 mins.

8. After the stain has set, get two paper tissue and place your slide on one of them. Put a coverslip on the slide (on top of the salivary glands). Fold the second tissue and place it on top of the coverslip.

9. Place your thumb on the over the coverslip and press down slowly and firmly. Use sufficient pressure but do not allow the coverslip or slide to slip or move.
12. Examine your stained, squashed salivary glands using the medium power objective lens. Look for nuclei and chromosomes. After you have located chromosomes, use the high power objective lens to see details of the chromosomes.

Gram Staining

Staining is an auxiliary technique  used in microscopic techniques used to enhance the clarity of the microscopic image. Stains and dyes are widely used in the   scientific field to highlight the structure of the biological  specimens, cells, tissues etc.
The Gram staining method was first described in 1844 by the Danish bacteriologist Hans Christian Gram, after whom the test was named.
The Gram staining test for bacteria is one of the most important tests in microbiology and is often one of the first tests performed in the identification of bacteria.

Gram staining is a differential staining technique that differentiates bacteria into  two  groups: gram-positives and gram-negatives. The procedure is based on the ability of microorganisms to retain  color of the stains  used during the  gram stain reaction. Gram-negative bacteria are decolorized by the alcohol,  losing  the  color of  the primary stain, purple. Gram-positive  bacteria  are  not  decolorized by alcohol and will remain as purple. After decolorization step, a counterstain is used to impart a pink color to the decolorized gram-negative organisms.

The mechanics of the Gram staining method is that the bacteria cell walls retain the crystal violet and subsequently added iodine, which complexes with the crystal violet, preventing the easy removal of the dyes. This step is known as the
“fixing the dye” step. During the subsequent addition of a decolorizer, a mixture of acetone and ethanol solvents, Gram-positive cell walls dehydrate, closing the pores in the cell wall, resulting in the retention of the crystal violet: iodine
complexes. In contrast, the decolorizer dissolves the higher lipid content of Gram-negative bacteria and the primary stain is able to leach into the solvent, essentially washing away the dye, leaving the Gram-negative bacteria unstained.

The length of the decolorization stage is critical as prolonged decolorizing will remove the primary stain from the Gram-positive cells and this will lead to false negatives during characterization of the microorganisms.

Finally, in order to visualize the unstained Gram-negative bacteria, a counter stain is added. Safranin, a basic stain that stains bacteria red. Some bacteria stain weakly with Safranin and the alternative counter stain Fuchsin is used.

Procedure for Gram's Staining
After the smear has been dried, heat-fixed, and cooled off, proceed as follows:

1. Place slide on staining rack and cover specimen with crystal violet. Let stand for 1 minute.
2. Wash briefly in tap water and shake off excess.
3. Cover specimen with iodine solution and let stand for 1 minute.
4. Wash with water and shake off excess.
5. Tilt slide at 45° angle and decolorize with the acetone-alcohol solution until the purple color stops running. Wash immediately with water and shake off excess.
6. Cover specimen with safranine and let stand for 1 minute.
7. Wash with water, shake off excess, and gently blot dry. The smear is now ready to be read. (Use oil immersion lens.)

Affinity Chromatography

Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. It enables the purification of a biomolecule on the basis of its biological function or individual chemical structure.
Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.
Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatographic matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Any component can be used as a ligand to purify its respective binding.
  
1. Affinity medium is equilibrated in binding buffer.
2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes
through the column.
3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Target protein is
collected in a purified, concentrated form.
4. Affinity medium is re-equilibrated with binding buffer.

G and R Banding

G-banding is a technique used to produce a visible karyotype by staining condensed chromosomes. It is useful for identifying various genetic diseases through the photographic representation of the entire chromosome complement. The metaphase chromosomes are treated with trypsin (to partially digest the chromosome) and stained with Giemsa. Dark bands that take up the stain are strongly A,T rich (gene poor). The reverse of G-bands is obtained in R-banding.

It is difficult to identify and group chromosomes based on simple staining because the uniform color of the structures makes it difficult to differentiate between the different chromosomes. 

Giemsa's solution is a mixture of methylene blue, eosin, and azure B. It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. Giemsa stain is used in Giemsa banding, commonly called G-banding, to stain chromosomes and often used to create an idiogram. It can identify chromosomal aberrations such as translocations and rearrangements.

limitations of this technique are the ineffectiveness of determining small translocations, detecting microdeletions, and characterizing the chromosomes of cell lines which are complex. However, it is a fast and low-cost technique to determine chromosome number, aneuploidy, large translocations, and macrodeletions.

A reverse Giemsa chromosome banding method that produces bands complementary to G-bands; induced by treatment with high temperature, low pH, or acridine orange staining.

Acridine orange was originally used to stain untreated chromosomes. Acridine orange (AO) is a base composition-independent fluorochrome that binds to DNA by intercalation and which gives relatively uniform fluorescence along the length of the chromosome arms. The dye binds very little to non-nucleic acid cell components, but it fluoresces orange-red when bound to single-stranded nucleic acids and yellow-green when bound to double-stranded nucleic acids. Following hot phosphate buffer treatment, R bands are yellow-green, and G/Q bands are orange-red. The major factor that contributes to R banding is the relative GC-richness of the R bands.

Phenol Chloroform Extraction for Plasmid Isolation

• Initial protocol same as Plasmid DNA isolation.
• After the wash with 75% ethanol, suspend the pellet in 500ul of TE buffer.
• Add equal amount of phenol:chloroform:isoamyl-alcohol (25:24:1), and centrifuge at 10,000rpm for 10mins.
• Three layers will be observed, carefully take the upper aqueous layer without disturbing the middle white layer.
• Transfer that to another eppendroff and add equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1), and centrifuge at 10,000rpm for 10mins.
• Again take the aqueous layer into another eppendroff and add equal amount of chloroform, centrifuge at 10,000rpm for 10mins.
• Remove the upper aqueous layer and add 1/30th volume of sodium acetate (pH5.2) and 0.7-0.8 volume of isopropanol.
• Centrifuge at 14,000 rmp for 30mins at 4 degree C.
• Carefully decant the supernatant, to the pellet add 100-200ul of 70% ethanol.
• Centrifuge at 14000 rpm for 10 mins, discard the supernatant and air dry the pellet.
• Add 50ul of TE buffer pH 8.