1. PCR .
2. Protein extraction and purification .
3. Protein concentration determination .
4. SDS-PAGE .
1. The aim of experiments :
2.1 The aim of PCR experiment is to replicate some DNA dimmers by using specific enzymes used for replication in vitro which is done in lab not by living organisms. 2.2 The aim of protein extraction and purification experiment is to extract some proteins and purify them by specific methods. 2.3 The aim of concentration determination experiment is to determine the concentration of proteins that extracted from the previous experiment. 2.4 The aim of SDS-PAGE is to separate proteins under denaturing conditions according to their number of subunits and size of those subunits. 2. Materials And Methods :
3.5 As the manual.
3.6 Take 5 grams of cauliflower + 25 ml of buffer extraction buffer ( PH Tris or NaCl) , but all other materials as the manual . 3.7 As the manual.
1. Find the small fractions that you froze at the end of the cell fractionation experiment.
2. Now, determine what volume of each sample will contain 50 µg of protein. For example, if you calculated that your protein yield was 5 mg protein /mL (5 µg/µL), you would need 10 µL of that fraction to give you a 50 µg concentration. Place the appropriate volume (based on protein concentration) of each sample into a labeled microfuge tube.
3. Add 1/4 volume of 4x Sample Buffer to each sample. For example, if the volume of your protein sample is 10 µL, add 2.5 µL of sample buffer. Also add 2.5 µL sample buffer per 10 µL of your protein standards.
4. Place the microfuge tubes containing your samples and sample buffer in a boiling water bath and boil the samples for about 5 minutes. Remove the microfuge tubes and place tubes on ice. Chilling the samples keeps them dense so that they “sink” when placed in the wells....