nothing is imposibble!

I finished my secondary education in SMK Taman Johor Jaya (1) which is ranked number 2 problematic school during my time, and climbed up to number 1 after I'm grad because my super juniors just burned the security guard pondok.

I was actively involved in St. John Ambulance Malaysia then and enjoying school politics as Duty Secretary of SJAM division SMKTJJ1.

Form 6, I have to changed to another secondary school, SMK Permas Jaya, which is a better managed school, but the students there are just too good, they will walk within the line, everyone uniforms are so neat, the toilet is so clean, nobody smoke, no fire burning, no ppl trying to steal the TV from library and the staff and head master are just too good and fair! make me not used to it!

The SJAM here is like a dead organization. With 300 members in the list. Nobody appeared on Saturday Ko-K activity. No proper activity. No proper AGM. As SJAM members, we decided to help them up.

After several meeting and discussion, I found out many things that I thk is possible, is impossible in their mind, esp from Physics class.

This is opposite of my idea that we should try before we say it is not doable, even we failed, we still can learned something from the failure, of course, we need to plan properly.

After so many years, many ppl still worry this and that, aiya, just do it la when u still young, dont scare fail ma...

However, after so many years, I also kena this virus, injected into my mind liao.

Is time to think again and adjust myself.

IF it is not possible, and if I'm not trying hard enough, I will be regret and SJAM of my form 6 school will not be revived.

Just remind myself not to be too EGO, a little bit can la... haha!

Is time to really think about it.

Colleages

Working with molecular biology, you need imagination, unless you got a microscopic eyes that can zoom into molecules. Here are some tools we using.

A spectrometry, to check on the concentration of bacterial cells, protein, as well as DNA by the reflextion of light.

An Autoclaving machine, heat up to 121C for 15mins, 15bar, in order to sterilize culture media, keep our tools from contamination by microbes.

A weight balance, to measure weight for chemicals, media powder.

Water distiller, to produce clean water free of contaminated chemical like nuclease and protease.

A water bath machine, maintain desired temperature for incubation.

A Polymerase Chain Reaction machine, (PCR), amplification of genes.

A UV light detector, to visualize the results of electrophoresis for analysis.

Kubota centrifugal unit, to separate cells, DNAs, proteins @ desired temperature.

Agarose gel electrophoresis, to run the gel in order to know what u get is what u want.

Not what u want? run again then, u just wasted another 60minutes.

There are a lot other useful stuff for molecular biology such as SDS P.A.G.E for protein analysis, crystallization wells and others.

Mistakes

Sometimes, feeling hopeless and demotivated when things go too slow...

1. E. coli Rosetta-gami2(DE3) pET32-phaCcs

not stable, the expression level keep reducing after sometimes.

Revival of glycerol stock and verification ate most of my times.

Production is still OK. Some part in my mind is asking me to retransform the plasmid again.

However current priority is still crystallization(produce protein fast), and my target for crystallization is end of MAY!

2. Own stupid mistakes(thinking not diverse enough)

Purification in July

The purified PhaC protein seems not pure enough, so, I'm thinking to purify the elution again using the regenerated resin and I did.

However, the results of the SDS PAGE analysis is not good, because the PhaC protein cannot bind efficiently to the resin.

I thought is because of the Regenerated resins. Hence, I try new resin (mistake: I missed something very important here when troubleshooting).

For the 3rd purification, I used the elution from the 2nd purification. The result was the same. Binding of the protein is not efficient. Cannot figure out, think and think and think, and finally....I'm feeling stupid.

There are imidazole with concentration of 250mM in the elution that i used for 2nd and 3rd purification!

My large-scale protein production end here. no more protein. Now speeding for protein production again. I want xstal!