Workshop: Day 2

Day 1 of the workshop was a success! Details of the same are here

After a wonderful kick-start of the workshop, we were all looking forward to the events of Day 2, and as expected, it was a great experience!

On 2nd June, 2016, we performed the following:

  • Fermentation of the transformed cells
  • Induction
  • Lysing of cells
  • Casting of SDS-PAGE gels

The summary of the day 2 events is as follows:

Day 1 had ended with plating of the cells on LB-Amp plates, followed by incubation. On Day 2, we could see colonies which meant that the transformation was complete. We were thrilled with our results, as this was a crucial part of the entire process. We scraped these cells from the plate and transferred them in LB-Amp broth, and kept the tubes for incubation. This will ensure the fermentation of the cells.

image
Anjali ma’am demonstrating transfer of culture from plate to broth

After the incubation period, we induced the cells using a lactose analog – IPTG inducer. The lactose analog ensures that our protein of interest gets expressed.image (1)

The inducing process is another crucial part, as it must be done at a particular stage when the cells are young and able to produce maximum amount of proteins.The induction process is then continued in the shaker for a few hours so that all the cells get induced and we get our desired protein.

This process helped us to understand the concept of induction better, and to do it practically was a very nice experience.

We then proceeded with lysing of the induced cells.

“Cell lysis refers to the disruption of the cell membrane and release of the cytoplasmic contents into the extracellular space.”

The induced cells were removed from the shaker and centrifuged at high speed to pellet the cells. We discarded the supernatant and resuspended the pellets, and then transferred it in a lager tube.

We then added a pinch of glass beads in the tube and lysed the cells by vortexing vigorously, followed by placing the tube on ice repeatedly to ensure the release of proteins from the cells.

We separated the lysate in a fresh eppendorf tube and allowed it to spin in the centrifuge. The supernatant was separated and collected in another eppendorf tube. The tube containing the pellet was resuspended, and both the eppendorf tubes (one containing the suspension and another with pellets) were then kept in the freezer overnight.

We then prepared the SDS-PAGE gels, which would be used on Day 3 of the workshop.

We had studied the principles of electrophoresis and SDS-PAGEin theory, but had never performed it. So doing it on our own gave us a better grab over the concept.

Setting of the SDS-PAGE system wasn’t very easy, as it required castingtwo different gels – resolving gel and stacking gel. The casting of the gels was done according to the protocol and the reagents used included buffers of different pH, acrylamide, polymerizing agents and a catalyst for the polymerization process. The reagents were added in different concentrations depending upon the type of gel.

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We first assembled the apparatus by properly placing the two glass plates separated by a spacer, and then sealed the three ends using agarose (to prevent the gel solutions from leaking out).

The resolving gel solution was prepared and poured first upto 70% height, and then a thin layer of water was applied on top of it. The gel was allowed to set for a while until it polymerizes completely. Post polymerization, we removed the water using a tissue paper. We then placed the comb depending on the depth of the well required. The stacking gel was then prepared and poured through the teeth of the comb till the top and around the teeth and allowed to set till it polymerizes.

image (2)
Anjali ma’am demonstrating casting of the gel

Once the gel had set, we kept it in the cooler to be used the next day. With this, day 2 of the workshop came to an end.

This was my first workshop and the overall experience was great! Not only did we get hands-on experience, but it also built up our confidence and put us at ease while handling the lab equipment. Anjali ma’am and her team were wonderful and helped us in each and every step. The explanation of every process and the principles of the reagents were explained, and our doubts were solved in an easy and simple way.

The workshop was a perfect combination of knowledge and fun, and it gave us a glimpse of how things are carried out in a R&D lab, or in an industry. I’m really looking forward to many more workshops!

Draft: Aanchal Udaynath

 

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