FYP

They say writing is a form of expression that takes the place of what can’t be said and it helps to organize thoughts.

At the beginning, we were tasked to look for mutation in gene p63 and p73 however; it was later switched to detecting polymorphism in gene p53 while the other team was to look for mutation in gene p53.

The possible polymorphism of p53 gene were identified at codon 72(Mildred’s target) and mine, codon 47. The wildtype of codon 47 nucleotide sequences is CCG (Pro) while the polymorphic codon 47 is TCG(Ser). This polymorphism has up to 5-fold decreased ability to induce apoptosis compared with the wildtype p53. The role of this polymorphism has to be researched even more indepth.

For now, we move on to the practical phase of our project which is to do a successful Polymerase Chain Reaction run and a good Gel Electrophoresis run.

The DNA samples for our PCR test were obtained by a method devised by Dr Alvin. Saline solution, 25ml were given out to each of us and we were told to gargle it and spit it back to the tubes. The disgusting saline solutions were centrifuge at 2500 rpm for 10mins? Pellets were observed and I think these were the DNA that sunk to the bottom of the tube and Dr Alvin sucked out the supernatant, leaving us with the pellet.

My sample was rejected because of food particles in it therefore; I have to rely on Mildred’s.

Out of the blue, a PCR 5x buffer (22ul) plus MgCl (6ul) was added to the tube where the pellet resides and then the entire content was transferred into an eppendorf tube which was later heated to 100 degree Celsius.

Shit, everything after this is hazy, I can’t recall and I forgot to note them down.

Supposedly the eppendorf tube filled with our DNA sample contains the buffer and salt. After boiling it, 1) we put into the freezer? 2) we pipette them into separate eppendorf tube and put into the freezer? 3) we pipette them into separate eppendorf tube with the buffer and salt previously prepared?

3 possibilities, which one is right? Anyone play socartic method with me?

Anyway moving on. The master mix were determined to be as followed

dNTPs : 1ul (Mixed, provided by Dr Alvin)
PCR grade water : 26/25ul (25 because 2ul of Tacq were used instead of 1 ul)
Tacq : 2ul
Primers : 2ul (We were left with gene specific primers as the other team had chosen all the RAPD primers)
PCR buffer : 15ul (Inclusive of 5ul of MgCl premix by us)
DNA template : 5ul (The ones we made and stored in the freeze in which some of the steps still eludes me)

Total reaction volume is 50ul

But we need a total of 4 reactions so its 200ul
DNA template needed should be 20ul therefore I can assume that I am right in the amount of buffer and salt solution added to the pellet in the first place but why 50ul pipette? I FORGOT.

The master mix is mixed with DNA template and Primers added in LAST, I forgot about the placement of the Tacq though, last as well? Gota find out more.

So, this was Tuesday work which involves in preparing our content for PCR and on Friday which is tomorrow, we have to do it fast.

Today, Thursday we did Gel electrophoresis.

Our PCRed products were supposed to be loaded with LOADING DYE and carefully inject into the well of the gel. Carelessly, I loaded them with the DNA MARKER. The errors were not rectified in time and thus, my gel became laddered. Well, that guy before me passed me the bottle and I’m in no mood to second guess anyone early in the morning but end up all of us got it wrong. That’s not the end of our mistake; Mildred loaded the sample, instead of the sample mixed with the dye and so, no bands shown.

I guess it is good that we made the mistakes today and not late in the fyp phase. I’ll be more careful and attentive in the future. Hopefully I’ll minimize all the forgetfulness and the mistakes.