Preparation for Cell Medium - CCB1 & CCB2

I worked with Namita today (February 13th, 2013) on the preparation of cell medium CCB1 (200ml) and CCB2 (50ml). We didn't make a lot of medium at once because we have to make sure that the medium is fresh every time (usually are kept 1 month at longest). The medium would prepare cells ready for transformation.

Materials

CCB1 (200ml)         CCB2 (50ml)
- Water                  - Water 32ml
- CaCl2                       - CaCl2
- MgSO4                   - glycerol 18ml
                              - MgSO4

Once bacteria are centrifuged, CCB1 "washes" cells to make sure there is not culture left. One the other hand, CCB2 is used to "store" those cells. Note that CCB2 contains glycerol, which is a very viscous liquid. We know that viscous liquids are harder to solidify under low temperature, and since cells are usually stored at -80C, glycerol's viscosity could prevent the cells / proteins from being damaged. The addition of CaCl2 and MgSO4 are to neutralize the negative charge of cell membrane.

Namita was very nice, and she demonstrated how to do each step and let me try CCB2 myself. First, I use a  25 ml pipette to pour 32 ml of DI water (filtered) into a flask. Next, I added 0.515g of CaCl2 into the water. After CaCl2 is well-dissolved, I used another pipette to add 18ml of glycerol into the solution. Namita told me that we don't want to suck up glycerol too fast because it may cause bubbles, reducing the accuracy. In molecular biology lab, we should really emphasize the accuracy and thoroughness of each step even if they seem basic, and this is a habit I should develop over time.

Before we added MgSO4, we need to autoclave the solution and other equipment to make sure they are not contaminate and to eliminate any errors. Autoclave is a device used to sterilize equipment and supplies by putting them under high pressure saturated steam at 121 °C for around 15–20 minutes depending on the size of the load and the contents. We put the caps loosely on the flasks and put a a short piece of autoclave tape on everything we want to autoclave. Once autoclaved, the stripes on the tape would turn black. Then, we took our cart downstairs to the autoclave room and autoclaved them for 30 min.

Meanwhile, we returned to Namita's office and we talked about some different biotechnology such as RT-PCR, microarray, RNAseq. They all are techniques one uses to determine what genes are expressed at a specific time. However, considering the cost and practicality, we do RT-PCR in this lab (will explain in next blog post!).

In the end, we went down to collect our sterilized equipment and medium, and we added 1M of MgSO4 to both medium. I really learned how careful and precise we should be in real lab and important steps before actually conducting an experiment, including labeling (name + dates) and sterilizing "everything", and I hope through more practices, I will eventually develop this good habit. Later, Namita will grow her cells, wash them and store them in the medium for the RT-PCR we are doing next week (so excited because I just learned it in class!).