Week 5 and Earlier Sharings

Good Morning, dear friends!

Okay, it's my turn to update all of you about my work at.. *ehem* somewhere in the west of Singapore. hehe..

TOPIC: Student Internship Programme

The Company

The company that I'm attached to is a bioinstrumentation start-up company. Just a few months old, it's a spin-off from one of Singapore's research institutes.

What is 'Bioinstrumentation'?

Well, from my understanding, it means instruments, or equipments, that are invented/developed to improve the works in the biology field, or more generally, in the laboratory.

So, my company has developed a technology that miniaturises cell culture and cell-based assays, both of which use smaller samples and take shorter time. *I would loVe to share with you how exactly this technology works, but I am not sure which information can be released.. and I haven't asked my supervisor. Hmm.. So, stay tuned, aite!*

I'm sure some of you know where my workplace is. And, I'd like to share with you something that I found interesting (sorry for the lack of a better word!):

Since many research institutes/pharmaceutical companies are situated here, the management has built a 'shared facilities', located in the basement. The first time I stepped into the area, the first thought that came to mind was, "Waaahh~ So clean, like hospital!" The floor's practically shiny, I tell you. Haha.. Okay, so, what are the services you can find there?

This place has many sections: supply center, media preparation (known as MediaPrep), washing and autoclaving service, confocal microscopy, flow cytometry (thanks, QJ, for enlighting us with the information *big smile!!*) and high content screening.

I like the supply center very much! How it works is that I make my order online (what kind of order, you'd ask? Let me see.. You can get all sorts of labware and reagents here from labcoat to alcohol-resistant marker to bleach to pipettes to petri dish to tube rack to polymerases to buffer solutions... and the list goes on~), and depending on the arrival time, I will go down from my laboratory to collect the items. Seriously, this integrated facility is efficient and saves everybody's time.

okay, NEXT!

My Jobscope

I do admin work as well as Research and Development (R&D).

In Admin, I make payment vouchers and purchase orders, write cheques as well as calling and emailing suppliers to ask for quotations or request for services. I'm also learning to use MYOB 'Mind Your Own Business', an accounting software.

In R&D, so far, I culture cells in flasks (plus changing medium and passaging). GoOd thing that I had learnt Mammalian Cell and Technology! However, since it had been a year ago, it took me some time (not to long, though!) to get the hang of it again.. cell culture technique, aseptic technique.. But now, yeah, I'm doing goOd =)

I also seed cells on the company's proprietary plates once the cells have reached 70%-80% confluence inside the culture flask. Haaa~ why seed cell on the plate? What's so special about this plate...? I'll tell you next time! What I can say for now is that the plate is FLAT and it's the size of the standard microscope slide. You'd think that the sample will slide off, right? right? Well, I'm going to let you imagine how cell seeding is done on a flat surface! *smile mischieviously*

Other than seeding, I also do aliquoting (of fluids, such as primary antibodies), clean CO₂ cylinderincubator and waterbath, store the labwares in the appropriate place, and help my colleague to key in inventory.

Since my company is very new, I observe how my colleagues work hard to improve on the product. Any problem(s) and complaint(s) from client(s), they will try their best to rectify and troubleshoot it. Additionally, some marketing stuff: like, you have to find distributors to market your product, locally and internationally, or you do 'demo' of your product to potential customers at medical/research institutes, companies and universities. Needless to say, there is a lot of work to be done if you are new and have a goOd product to offer the scientific community.

Oh! I've just remembered: My laboratory is small, slightly bigger than our tutorial room, so, there aren't separate rooms or partitioned sections for washing, preparation and cell-culturing. Boxes of newly-arrived items or equipments have to be stored or put aside to minimise obstruction in the room. Fortunately, the essential equipments for cell-culturing work (laminar flow hood, inverted microscope and CO₂ cylinder) are positioned near to each other.

Okay, I guess that is all I have for now. I would have liked to post protocols and other 'scientific knowledge', but I've yet to gain one or two that's worth sharing with all of you. Still searching.. and still learning.

^_^

Till we meet again,
Nor Liyana bte Noor Mohammad
0607927A
TG02 - Group 8

Basic Introduction to Flow Cytometry

Hi guys, it’s the 5th week already, everyone still going great? Hope you are =)

Anyway, it’s not my turn to post this week, but I decided to still post something this week so to help everyone understand more about the principle of Flow Cytometry which I am fortunate enough to be working with one.

Why would I want to do this, is because I am worried if everyone understand what I was posting during my 1st week where I bombarded everyone with tons of information.

First, over the weeks, I am going to introduction on the following basic principles:
1) Hydrodynamic focusing of the sample
2) Light collection & how they are measured
3) Sorting principle of Fluorescent Activated Cell Sorter (FACS)
4) Data analysis for flow cytometry

For this posting, I will only be explaining about hydrodynamic focusing of the sample:

I am sure all of you know that Flow Cytometry is used to analysis the cells profile based on varying factors (I’ll touch more about the factors under point 2 for next posting – refer to above).

However, before the data could be obtained, the cells have to first be loaded into the Flow Cytometry for sample processing. How the sample is load into the Flow Cytometry is very important, as the sample stream has to be in single cell line in order to record single cell profiles.

Figure 1: Cross-sectioned laser beam focused by laser optics lens
Retrived, July 25, from:
http://biology.berkeley.edu/crl/flow_cytometry_basic.html

The above diagram shows how the sample is being focused into a single cell stream before passing through the laser beam, which is the interrogation point where data about the cells are collected. As you can see, the flowing sheath fluid cause the focusing of the sample stream into single cell line.

Hydrodynamic focusing:
A higher pressure from the flowing sheath fluid causes the lower pressure from the sample to form a single flowing cell line.

That is why the surrounding sheath fluid has to be higher in pressure than the sample fluid, else hydrodynamic focusing could not occur.

That’s all for this posting, will post the next topic the following week.

Please do tell me if you find this useful for you in understanding, as this is something extra I plan on blogging to help everyone understand Flow Cytometry. Because I worried that my first post was too much in the content and hard for everyone to understand.

So, is it okay if I continue the postings to explain the basic principles of Flow Cytometry?

Everyone! Have a nice day =)

Posted by:
Low Quan Jun (0607243C)
TG02
Group 08
25 July 2008

Week 4 ( OMG 1 month already! ) :)

Hey dearest course mates! Hope all of you are doing fine. It’s already been a month and I miss you guys and TP so much! ((:

Okay, besides those irrelevant things, I am attached to a research company together with Lyn, so our information might be similar.
My company is focusing on Pharmacogenetics. Now, you might be asking what is Pharmacogenetics? Is it the combination of Pharmacology and Molecular Biology/Genetics?

The answer is yes. Basically, pharmacogenetics is the study of genetic variations (polymorphisms) in individuals that bring about a different response to the same drug. Thus, for example, person A might take the drug and benefitted from it, whereas person B is administered with the same drug, but suffered from adverse drug reaction.

The 3 main areas that are extensively studied for their genetic variations involves gene that codes for 1) receptors, 2) transporters and 3) drug metabolising enzyme. As we have studied previously in Pharmacology, drugs need to bind to receptors, need to be transported to a specific target/ receptor and also need to be metabolized by certain enzymes. Thus, if it happens to be that the genetic variation in an individual is the one affecting the receptor, transporter or metabolism enzyme, then, the person would show a different pharmacodynamic and pharmacokinetic response.

Okay let’s recall! Pharmacodynamics is the study of what the drug does to the body while pharmacokinetics is the study of what the body does to the drug (absorption, distribution, metabolism and excretion). Thus, with that, the person would either have a lack of an efficacious response or more side effects.

Now, do you know why the same medicine might work very well for your friend (eg) and not for you, or vice versa? :)

To summarise what I am suppose to carry out for my research, my main objective is to find out the genetic polymorphism of a certain transporter gene, between 3 different ethnics group: Chinese, Malay and Indians and detect significant polymorphism of the drug that might affect its transportation factors, thus affecting the drug level in the body. The type of polymorphism that I am suppose to find out on, is SNPs, which is a single nucleotide polymorphism.

What is SNP? It is a single base (A, T, G and C) change that is found throughout the genome. For eg ATT GCC TCA to ATT GCC TAA. So, how does one nucleotide/base affect the gene? Because 3 nucleotides make up a certain codon that expresses for a certain protein, a single base change would change the codon and will result in the expression of a different protein. However, not all SNPs are significant, as it is common among individuals and does not affect the expression of proteins. Thus, for those SNPs that are significant, we can detect it by analysing the gene sequence carrying the SNPs. This will be explained in further details in later post.

As for now, I would describe the first step that is needed to be carried out, that is primer optimization using test DNA.

Primer optimization

Primer optimization is done to find the appropriate optimum annealing temperatures (temperature at which primer binds to DNA) that is needed later during the PCR (polymerase chain reaction) to make multiple copies of specific target sequence to be analysed.
As most primers’ annealing temperature is between 55-65oC and most appropriate number of cycles is 30, I can start testing the primers at that temperature and cycle number.

Methods

1. Prepare a master mix of 13 times the stated amount (need only 12 tubes but 13 times to overcome pipetting errors)

(total: 12 tubes)

Component(s): H20, 10x Buffer, MgCl2 (25mM), dNTPs (10mM), Forward Primer (10µM), Reverse Primer (10µM), Taq polymerase, Test DNA

Total Sample x 1 (µL): 6.9, 1, 0.6, 0.3, 0.2, 0.2, 0.3, 0.5 respectively. Total: 10µL

Sample x 13 (µL): 89.7, 13, 7.8, 3.9, 2.6, 2.6, 3.9, 6.5 respectively. Total: 130µL

[ Sorry I had to put it this way. Blogger doesnt allow to put table, right? I cant seem to copy and paste my table from word. :( ]

2. Prepare an ice-box.
3. Collect the different reagents and place them in the icebox. (Taq polymerase is to be collected last after adding all the other reagents as it will denature at room temperature or less than 20 degree Celsius if it is removed for too long)
4. Prepare a strip of 12 tubes and label them 1-12
5. Add in all the reagents into the master mix tube.
6. Place the remaining reagents back into the respective fridges.
7. Spin the master mix tube down for a few seconds to ensure all reagent is mixed
8. Pipette 10µL from the master mix into each of the 12 tubes.
9. Spin down the tubes again for a few seconds, to also remove all air bubbles.
10. Place the 12 tubes into the Thermogradient (thermocycler)
11. Place 4 empty tubes at each of the 4 corners to balance it.
12. Programme it at 55-65oC and carry out for 30 cycles.

Conditions of Thermogradient

Step 1: 95oC x 3min
Step 2: 94oC x 45s
Step 3: 55-65oC x 45s
Step 4: 72oC x 45oC
Step 5: Repeat steps 2-5 x 29 (total 30 cycles)
Step 6: 72oC x 7min
Step 7: 15oC x Infinity
-End-

This reaction takes about 1 and a half hours, so to not waste time, agarose gel is to be made (2nd step). I will explain about it in my next entry so my post wouldn’t be too lengthy ya, and before I bore you guys further. :)

So this is it, any queries, feel free to ask, I won’t bite. Hehe. Take good care of yourself and I’ll see you soon on the 25th!

Best regards,
Liyanah Zaffre
0607718D
TG02