Accuzyme PCR and Gel Preparation

Last Wednesday (February, 20, 2013) Namita explained that since some of the RNA they had extracted was not dissolving into water, they would probably save the RT-PCR until next week. Meanwhile, I did a PCR and prepared some gels with her:)

Accuzyme PCR is a kind of PCR that uses accuzymes as DNA polymerase. Depending on the enzymes we use, we would need to "slightly" adjust the temperature in each process (denaturation, annealing, elongation). For example, the optimized temperature during elongation for Taq polymerase is usually 72C while 70C for accuzyme. One of the benefits of accuzyme is its high fidelity (lowering the errors in replication).

Materials (for 50μl)

• 2x Accuzyme Mix               25μl
• MgCl2                                  1μl
• ddWater                             20μl
• primers (10mM)                    1μl each (forward + reverse)
• templates

We labeled 3 microtubes:  primers mix, reaction mix 1 and reaction mix 2. Accuzyme Mix 2x already contains buffer, accuzyme, and dNTP we would need for PCR. Moreover, we need to dilute the primers mix from 100mM to 10mM. In convention, we would add the solution that has the  the greatest volume first, but in this case we added water first because we want to add the enzyme the last to avoid protein denaturation. Yet, to make sure the concentration of the accuzyme mix is consistent, it's better to wait the protein solution to become completely liquid before adding to our reaction mix.

Then, we put the reaction mix into thermal cycler where PCR is processed.

Thermal Cycler (Bio-Rad)

The major 3 processes of PCR is actually subdivided into 5 process:

1. initial denaturation (2-3 min)
2. denaturation (15-30 sec)
3. annealing  (15-30 sec)
4. extension (1.5-2 min/kb)
5. final extension (7min)

Step 2, 3, 4 go through about 34 cycles before going into final extension (to make sure the nucleotides are fully replicated). We put in the temperature and time we want during each step into autowriter. The whole process lasts about 2 hrs.

An example of  the screen of autowriter

Meanwhile, we prepared some electrophoresis gel. First, we pour some buffer in a flask. Then, depending on what concentration of gel you want to make, add the according amount of agarose into the buffer. The higher the concentration, the more selective the gel is, and the better for small molecules. In this case we made 0.8% and 1.2% gel. Next, we put the flasks into a microwave for 2-3 min to well-mix the solutions while also sterilize them. Once the solutions cooled down to 60C we add ethidium bromide (EtBr) - a highly mutagenic and moderately toxic (radioactive) chemical that is commonly used for DNA staining  in electrophoresis. Thus, we must wear gloves when doing this. Last, load the buffer into models with combs, wait for them to solidify and you are done!

In the end, I observed some group member talking about the RNA for RT-PCR I mentioned previously. They thought there might be some contamination that barred RNA from dissolving. Nonetheless, they decided to use the gels we just made to check whether their data is reliable.

We did quite a few things today! By using PCR we can make many recombinant plasmids for bacterial transformation and let the bacteria to produce the desired molecules. I also learned how to do PCR, so next time I would be able to do it on my own:) I feel great when I learn new things, and I hope that the more I learn, the more helpful I could be:)

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!).

Optical Density

Last Wednesday (February 6, 2013) I worked with Eun Ji. We didn't do much because she was very busy and had already done her experiment before hands.

At 2 pm, we attended a mandatory safety education at one of the largest laboratory because a few weeks ago an undergraduate was in a lab alone and for some reason hurt himself / herself quite severely. They demonstrated the emergency eye wash and shower. They also invited some people to try pulling the "pole" (I don't know how to describe it) so we can get familiar with the force. It's actually really fun because it was my first time to see the shower on! In the end, they really emphasizes that safety is more important than flooding the laboratory.

Later, Eun Ji showed me how to measure optical density of bacteria. Optical density (OD) is the measure of the amount of light absorbed by a suspension of bacterial cells or a solution of an organic molecule with the use of a spectrophotometer. The more bacteria in a solution, the higher the OD. Eun Ji started to grow her bacteria the day before and measure its OD every 2hrs. Each type of bacteria has the greatest protein production efficiency at a certain OD, in this case at OD = 0.5. Thus, when Eun Ji's bacteria reaches OD = 0.5, she will inject a reagent, and the bacteria will start to make the protein she wants. The last time she checked, the OD was still 0.4, so she hypothesize  it would reach 0.5 at 2 pm.

However, when we checked it, the OD has already exceeded 0.8!! Eung Ji explained that it's because the bacteria had now reached the exponential phase (2nd), in which they multiply very rapidly. I thought we had to start the whole experiment all over again, but luckily all we need to do is to dilute it:)

So we went back to the original lab to dilute the solution. However, doubling the volume does not necessary means lowering OD by half, so we can only do some trial-and error. We really need to well-mix the solution when adding new medium, so she keeps pouring back and forth the solutions (though she said ideally we would use pipettes, but for the sake of time...).

The middle two have bacteria (milky), and the outer two are the new medium we want to add to the original solution to dilute them.

The second time we got 0.65, still too high. We ending up adding about 200ml of medium, and the OD was successfully reduced to 0.53!

OD measured by spectrophotometer; OD = 0.532

Eun Ji then added some reagents to the solution and took them down to a cell growth room where the cell incubator shaker was set in 30C. Then, she will have to check it 6 hrs later. Eventually, she will break the bacteria and collect the desired protein.

It was interesting to see how much process one has to do just to get a desired OD although I didn't get to touch anything because Eun Ji said that type of E.coli. can, but rarely, cause diarrhea, which is also why she used alcohol to wipe the flask every time she opened it. We ended early because she has a group meeting at 3:30, so I spent the last half hour doing my work. Next week I will be working with Namita, and I am looking forward what we are going to do together:)