Day 14

Day 14 - Panic

In the morning, I showed my PCR result to ZD and he just said "Congratulation". So after that, I proceed with TA cloning. TA cloning is the whole process of cloning DNA using the advantage of the overhang A that was produced by the taq polymerase during the PCR reaction. In the TA cloning, the insert (PCR product) has overhang A while the vector contains an overhang T. Thus, they can form complementary base pair which make it easier for ligation.

At first, I misinterpret ZD instruction. I purified my PCR product without leaving any for TA cloning. In the middle of purification process, suddenly ZD just told me that if the purification process was not successful, I have to repeat the PCR. I felt so stupid and so stressed because of that. Luckily the gel run showed that my purification process was successful... Fiuh....

For ligation, instead of using ligase, we use topoisomerase. I don't really understand the whole process but it's obviously much shorter and simpler than using ligase. After ligation, we proceed with transformation but I had to wait until ZD finished preparing the competent cell. He told me that the competent cell that SQ, FM and I prepared last time was contaminated and "not good". So he prepared new competent cells.

After transformation, I just spread the transformed bacteria into a LB plate with IPTG and wait until the next day to select the colonies that has been successfully transformed.