this is very helpful presentation for using different lab techniques in biotechnlogical field.....you can download and enjoy this...thank you

1. Basic Labe Molecular Techniques Used in Biotechnology
.
Muhammad Kamran
1457-315026
MSc Biology
Semester 5th
Project Presentation

2. Basic Labe Molecular Techniques Used in Biotechnology

3. Plant tissue culture

4. MATERIAL AND METHODS

5. Steps involved in the invitro
micropropagation
Cleaning of glassware
Preparation of nutrient medium
Selection and sterilization of explant.
Inoculation of aseptic explant in to nutrient medium.
Proliferation of shoots on a multiplication medium.
Transfer of shoots for sub-culturing.
Rooting and hardening of plantlets
Field trials.

6. Steps
.

7. RESULTS AND DISCUSSION
 After the first four weeks of culturing, the external leaf
primordia of explants turned green which were initially
creamy white. The size of the explant also increased,
while blackening was observed at the base of the explant.
This blackening of the explant may be due to secretion of
phenolic compounds.
 From the results it was also observed that the cultures
showing higher rate of multiplication in the first two or
three subcultures continue this behavior in the next
subculturings. Therefore, from the commercial point of
view, the explants Showing higher rate of multiplication
initially may be continued in the coming period. Cultures
showing potential for initially low rate of multiplication
may be discarded in the beginning to avoid the wastage of
time, space and other resources.

8. PCR

9. Steps

10. DNA EXTRACTION
 DNA isolation is a process of purification of DNA from sample
using a combination of physical and chemical methods. The first
isolation of DNA was done in 1869 by Friedrich Miescher.
 DNA extraction by CTAB method
 Crush 2-3 leaf samples in mortar and pestle with liquid nitrogen (-
196 0C).
 Add 2-3 ml of pre-warmed 2X CTAB having 0.05% B-marcepto-
ethanol. (50 µl β-mercaptoethanol / 50 mL 2X CTAB Buffer use
within 2-3 days).
 o Transfer 750 µl of sample extract to eppendorf tube (1.5 ml)
 o Incubate the eppendorf tubes at 65 ºC for 30 minutes.
 Add 0.8 volume of Chloroform: Iso-amylalcohol (24:1) solution to
the eppendorf tubes and mix gently.
 o Centrifuge the samples at 14000 rpm for 10 minutes.
 Transfer the supernatant to a new tube and add equal volume of
pre-chilled Iso-propanol and mix gently by inverting the tubes. Also
80 µl of 3M sodium acetate to adjust ion balance.

12. ELISA

14. ELISA Results
 The ELISA assay yields three different types of data output:
 1) Quantitative:
 ELISA data can be interpreted in comparison to a standard curve (a serial
dilution of a known, purified antigen) in order to precisely calculate the
concentrations of antigen in various samples.
 2) Qualitative:
 ELISAs can also be used to achieve a yes or no answer indicating whether
a particular antigen is present in a sample, as compared to a blank well
containing no antigen or an unrelated control antigen.
 3) Semi-quantitative:
 ELISAs can be used to compare the relative levels of antigen in assay
samples, since the intensity of signal will vary directly with antigen
concentration.
 ELISA data is typically graphed with optical density vs log concentration to
produce a sigmoidal curve as shown in Figure . Known concentrations of
antigen are used to produce a standard curve and then this data is used to
measure the concentration of unknown samples by comparison to the
linear portion of the standard curve. This can be done directly on the graph
or with curve fitting software which is typically found on ELISA plate

15. ELISA Data Graph optical density vs log concentration