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:)