Day 2 of the workshop was a great experience as said earlier. The PCR technique and restriction enzymes which we learnt theoretically; doing it practically was amazing. We were very excited for day 2, because we have been just hearing about “PAGE” a lot but never had seen or done it. The extraction of the genomic DNA was another interesting experiment. You can find the details here!
We started our day 3 with SDS PAGE experiment first.
The separation of macromolecules in an electric field is called electrophoresis. A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins. The method is called sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The setup of the SDS PAGE instrument wasn’t that easy as it was for the normal gel electrophoresis.
Regardless of the system, preparation requires casting two different layers of acrylamide between glass plates. The lower layer (separating, or resolving, gel) is responsible for actually separating polypeptides by size. The upper layer (stacking gel) includes the sample wells. It is designed to sweep up proteins in a sample between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when they reach the separating gel.
As casting was done, we were supposed to make our protein sample by just adding the
required amount of dyes to the already prepared samples. These samples were then carefully loaded in the gels using micropipettes. The gel was then run.
When the dye run was completely at the bottom of the casted gel, it was time to stop the run.
The gels were carefully taken off from the instrument and then stained with coomasie blue. The gel takes up the whole stain temporarily whereas the stain sticks to the proteins permanently.
Later on the gels were destained and the protein bands could be actually seen. This can be further analysed. The beauty of this gel is that it can be stored for a longer time after destaining it.
The next experiment we performed was extraction of Genomic DNA.
We were going to extract Genomic DNA from onions.
We crushed the onions using a mortar and a pestle. The crushed onions which had a paste like consistency was treated with a detergent solution which had chemicals to carry out cell lysis. We kept the whole solution in the water bath at 60 degrees Celsius and immediately cooled for 5 mins to ensure complete cell lysis
Then we filtered out the extract using a muslin cloth. Then 600 microlitres of extract was taken in an eppendorff tube. 900 microlitres 70% chilled ethanol was then added to the 600 microlitres of extract and centrifuged. The supernatant was discarded and the ethanol which was excess was evaporated. After this, TAE was added and the pellet in the eppendorff was solubilized completely using hot water bath or just shaking.
This clear solution was then taken for running optical density using a UV VISIBLE SPECTROPHOTOMETER. The ratio of absorbance at 260nm and 280nm was taken. This ratio is supposed to be 1.8 ideally. The ratios observed are compared to the ideal ratio and the purity is concluded accordingly.
The experience of these experiments was very good. Handling the micropipettes which we always wanted to do was good. Handling those micro quantities, getting PCR results when you do it for the very first time, team work was really great. The principles behind each reagent and how the experiment actually works was well explained by the teachers. It was a very great experience as an undergraduate student. Looking forward for more such exciting workshops!