What type of practical work done in Pakistan's labs?

 

DNA EXTRACTION:

Principle:

The extraction of DNA involve three major steps

1.      Cell lysis

2.      Protein separation

3.      DNA purification

Procedure:

1. First add 1ml TE buffer into 200ul blood and mix it very well.

2. Centrifuge at 4000rpm

3. Discard the supernatant and add900 ul TE buffer.

4. Centrifuge at 4000rpm.

5. Discard the supernatant and add 800 ul TE buffer.

6. Centrifuge at 4000 rpm.

7. Discard the supernatant and add 200ul TEN buffer, 20ul SDS and 10ul proteinase-k.

8. Incubate at 56 degree.

9. Place the tubes on ice and add 6M nacl.

10. Centrifuge at 4000 rpm for 15 min.

11. Transfer the supernatant in a freshly labeled tubes.

12. Add equal amount of chilled isopropanol.

13.

Centrifuge at 8000 rpm.

14. Add 200ul absolute ethanol

15. Centrifuge at 8000 rpm.

16. Add 200ul ethanol.

17. Centrifuge at 8000 rpm.

18. Discard the supernatant and add 100ul Low TE buffer.

19. Store DNA.

Result:

Presence of DNA can be confirmed by electrophoresing on an agarose gel containing ethidium bromide.

PCR:

Principle:

 

The PCR technique is based on the enzymatic replication of DNA. This PCR uses primer mediated enzymes for the amplification of short segment of DNA.

Procedure:

PCR procedure:

Standard PCR consists of four steps

1.       Add required reagents or mix and template to PCR tubes.

2.       Mix and centrifuge.

3.       Add mineral oil to prevent evaporation in a thermal cycler without a heated lid.

4.       Amplify per thermal cycler.

5.       Evaluate amplified DNA by agarose gel electrophoresis followed by ethidium bromide.

Result:

PCR tests are highly accurate way to diagnose certain infectious disease and genetic changes. It is the test to detect genetic material from specific organism, such as virus.

Staining:

Principle:

Simple staining involves directly staining the bacterial cell with a positively charged dye in order to see bacterial detail, in contrast to negative staining where the bacteria remain unstained against a dark background.

Procedure:

1.     Use a inoculating loop to transfer the bacterial containing suspension.

2.     Disperse the bacteria on the loop with the drop of distilled water.

3.     Allow the smear to dry.

4.     Then heat the smear through Bunsen burner.

5.     Cover the smear with methylene blue and allow to dry.

6.     With the help of water wash the slide to wash off excess methylene blue.

7.     Saturate the smear with the help of iodine.

8.     Then wash the excess iodine with water. Place the stained smear under the microscope and observe the smear.

 

Result:

If bacteria remain purple they are gram positive, if bacteria remain red they ate negative bacteria.

Quantitative and qualitative result of Carbohydrates:

Carbohydrates test are taken to differentiate the monosaccharide from disaccharides and polysaccharides.

1.     Molisch test:

Principle:

Dehydration of sulfuric acid into furfural.

Procedure:

Take 2ml of carbohydrates into a test tube and take two drop of molisch reagent. Mix well and add 2ml of sulfuric acid.

Result:

Appearance of purple color ring is positive molisch test.

2.     Benedict’s test:

Peinciple

Carbohydrates with free aldehydes group have the ability to redure solution of various group.

Procedure:

Take 5ml of Benedict’s reagent and add 8 drops of carbohydrates .Boil over flame.

Result:

Benedict’s test is semi-quantitative test.

3.     Iodine test:

Principle:

This is test for polysaccharides.

Procedure:

Take 1ml of given sample in test tube and add 1ml of distilled water and add 2 to 3 drops of lugol’s solution. Observe the color.

Result:

            Appearance of purple color show positive test.

 

Ninhydrin Test:

 

Principle:

Ninhydrin reacts with the amino acid of the free amino acid in the test sample and oxidize the compound.Ninhydin react as oxidizing agent.

 

Procedure:

Add here drops of ninhydrin solution in ethanol and heat the solution for five minute.

 

Result:

 

A positive test is indicated by the formation of red color.

 

Biuret Test:

 

Principe:

The biurets test for proteins positively identifies the presence of protein in solutions in with a deep violet.

 

Principle:

Take clean test tube.

Add 1 -2 ml of test solution.

Add 2ml of biuret reagent

Shake well and observe color.

Result:

Purple color appearance show positive test.