Some Tips for Salivary Gland Dissection and Preparing Nice Slides

• Reagend 4 should be at least 50 ml although 60 ml is better to not let the coverslip stick to the slide while swirling it.
• Reagend 4 should not be less than 50 ml, otherwise there will be air bubbles at the edges of the coverslip which will result in crystalization of the sample because of vaporization of acetic acid.
• Reagend 2 and 3 should be fresh meaning at the end of each hour they should be reprepared. Each 30 ml of these reagends should also be changed after 10 minute.
• Reagend 3 and 4 should be kept their caps closed because not to let acetic acid to evaporate.
• Before swirling the coverslip on the slide, you should press gently on where the salivary glands are. This is to make nucleus come out of the cells and stick to the slide.
• Swirling makes the chromosomes spreaded on the glass slide. Therefore to have nicely spreaded samples you should not afraid to push while swirling. After some time of experience the optimum force will be found.
• Salivary glands should not be sepereated from each other and they should be placed at the center of the slide in the reagend 4. This is for having a big number of chromosomes in a single slide.
• The edges of the coverslips should be marked to know where is the sample on the slide. This will help during primary and secondary antibody staining. 50 ml of primary and secondary antibody solutions will be enough for one slide.
• The slide and the coverslip should be cleaned just before dissection and must be dust free.
• The last pushing step after turning the slide upside down is for to make chromosomes stick to the slide.
• After placing the slide in nitrogen coverslip should be removed immediately and fastly (in one shot) with scalpel.
• Slides might be kept in PBST at 4°C overnight.
• Reagends 2 and 3 can be used for max 2-3 hours. Always prepare fresh is dissecting for longer time.
• Reagends 2 and 3 could be prepared in large volumes without adding paraformaldehyde. Just before using small volumes can be prepared by adding proper volume of 16% paraformaldehyde.
• The best is to keep animals on 18°C, the salivary glands will be better developed.
• Starting from L2 stage larvae shoul be fed with yeast.
• The tubes should not be very crawded to let larvae develop nicely.
• Salivary glands that are balloon like and transparent shouldn’t be processed. They will not give nicely spreaded chromosomes.
• To have nice salivary glands and nice slides with well spreaded chromosomes, moving larvaes should be selected.

Salivary Gland Dissection

Reagend 1:  28 mikrol of 10% NP-40

                   50 mikrol 10X PBS

                   422 mikrol ddH₂O        

Reagend 2:  40 mikrol 10X PBS

                   100 mikrol 16% paraformaldehyde

                   80 mikrol 10% NP-40

                  180 mikrol ddH₂O

Reagend 3: 180 mikrol of 100% acetic acid

                   120 mikrol ddH2O

                   100 mikrol 16% paraformaldehyde

Reagend 4:  450 mikrol of 100% acetic acid

                   550 mikrol ddH₂O

Dissection:

Dissect salivary glands from wondering L3 larvae in 30 mikrol of Reagend 1.

Remove fat body from the glands.

Transfer the glands to mikrol ml Reagend 2, incubate for 40 seconds.

Transfer the glands to mikrol ml Reagend 3, incubate for 40 seconds.

Transfer the glands to 50 mikrol Reagend 4, that is placed in a slide, incubate for 1 min.

Cover with cover slip, gently push with a finger and move in a circle.

Turn the slide upside down, place in a layer of paper tissue, push vigorously.

Place the slide in liquid nitrogen and remove cover slip with scalpel.

Incubate slide in PBST (PBS + 0.1% Tween20)

Banding patterns and Polytene Chromosomes

Special chromosome staining procedures have revealed intricate sets of bands (transverse stripes) in many different organisms. The positions and sizes of the bands are highly chromosome-specific; therefore, they represent useful landmarks. There are Q bands (produced by quinacrine hydrochloride), G bands (produced by Giemsa stain), and R bands (produced by reversed Giemsa).

A rather specialized kind of banding occurs in a few organisms whose chromosomes can replicate their DNA many times without separating. This produces giant chromosomes, which are in essence magnified versions of the unreplicated forms. Consequently, the natural banding patterns of the chromosomes become readily visible and can serve as landmarks. These polytene chromosomes (polytene means “many-stranded”) are found in highly specialized cells of Malpighian tubules, rectum, gut, footpads, and salivary glands of the dipteran insects such as houseflies, mosquitoes, and fruit flies.

The fruit fly Drosophila melanogaster is a much-studied example. This insect (a diploid) has a 2n number of 8, and these eight chromosomes are present in most cells. However, in the cells of the special organs that contain the polytene chromosomes we see some interesting peculiarities. First, there are only four polytene chromosomes per cell (not eight) because during the specialized replication process the members of each homologous pair unexpectedly unite with each other. Second, all four polytene chromosomes become joined at a structure called the chromocenter, which is a coalescence of the heterochromatic areas around the centromeres of all four chromosomes. The chromocenter of Drosophila salivary gland chromosomes is shown in Figure, where L and R stand for arbitrarily assigned left and right arms.

Along the length of a polytene chromosome, there are transverse bands. Polytene bands are much more numerous than Q, G, or R bands, numbering in the hundreds on each chromosome. The bands differ in width and appearance, so that the banding pattern of each chromosome is unique and characteristic of that chromosome.

Molecular studies have shown that in any chromosomal region of Drosophila there are more genes than there are polytene bands, so the bands do not represent genes. Similarly, the significance of the Q, G, and R patterns in other eukaryotic cells is not clear. They probably reflect the degree of compactness of the DNA, but it is not known how this pattern is maintained.

http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mga&part=A154

have fun with your drosoflies :D

If you are bored,

let your flies help you have fun :D

hints about plasmid digestion

APE is a very useful tool for selection of required restriction enzyme.

***

Protein contamination in the plasmid DNA solution doesn't supposed to effect the restriction reaction (though A260/280 is better to be higher than 1.8).

***

TE Buffer is not recommended as an elution buffer for plasmid DNA that will be digested, although it provides a long time storage conditions by chelating the Mg+2 ions from the DNA (Mg+2 and other ions makes the DNA more prone to degradation). 

***  

After the digestion reaction completed,  oncentration of the loaded volume should be calculated and it shouldn't be more than 75 ng/ul. If there is very concentrated plasmid in the wells, linearized DNA will run slower than expected. 

 ***

Molecular Biology Vector Sequence Database is a useful link for whole sequences of several vectors. 

***

Search thousands of genes, chemicals, pathologies and much more...

http://www.wikigenes.org/

From the first of Morgan's mutants to polytene chromosome squashes to the early studies of patterned embryonic gene expression, Drosophila genetics has been a strongly visual field. The power of exciting pictures to capture attention (the four-winged fly), to introduce innovative new tools for analysis (GFP-exu) or illustrate a fundamental principle (the Bicoid gradient) has been a driving force for the research community and has drawn new scientists to Drosophila research. Such images are not limited to photographs, as illustrated by the activity charts of per mutants and the homeobox 'zooblot'. What all these images have in common, in addition to the pride taken by the creators in generating the data, is the encapsulation of an important biological result in a striking visual image.

In recognition of this fact, the Drosophila Board, supported by the Genetics Society of America, has initiated an annual award to recognize, celebrate, and encourage the continued generation of compelling images that communicate important biological results relevant to Drosophila.

http://www.drosophila-images.org/