Sunday, July 27, 2008

Week 5 SIP/MP Experience

Hello all, its been 5 since SIP/MP started and it was great seeing you all on friday again. This week it is my turn to share my SIP/MP experience to you all.

I am attached to a plant biotechnology lab, and under the TSO, I help in making plant media. I make two kinds of plant media. One has activated charcoal in it and one does not. The steps to making the plant media are similar, therefore I shall explain one, and list the constituents of the other.

Preperation of tissue plant media
1.) Fill up two 3Litre beakers to over 2.5L with MilliQ water.*
2.) Place the beakers on seperate magnetic stirrers.
3.) Add the constituents in the following order after weighing them/determining their volume in each beaker. (MS media formulation (Murashige and Skoog)-6.45g; Myo-30ml; Organic elements-30ml sucrose 60g).*
4.) Top up the water until both beakers are 3L full.
5.) Next the pH is adjusted to a range of 5.2 to 5.21 using the pH meter
6.) Activated charcoal(1.5g) is added next and allowed to be distributed in the media.**
7.) Next 30 vitro vents(containers that will carry the media) are prepared.
8.) 1.6grams of agar powder are then poured into each vitro vent.
9.) Next a marked measuring cylinder specially used to measure for the volume needed for each vitro vent is used and the media is poured to fill up all the 30 vitro vents.
10.) The vitro vents are then covered with a cover and autoclaved.

Constituents for second set of plant media
1.) MS 6.45g
2.) Myo 30ml
3.) Organic Elements
4.) Sucrose 30g
5.) pH 5.2-5.21
30 vitro vents
1.6g agar

*Avoid parallax error by placing the eye onthe same level as the meniscus and read the bottom of the meniscus
**The activated charcoal does not dissolve into the media, therefore it must be thoroughly in the media before it is poured into the vitro vents. And after half the beaker is emptied, place the beaker back on the magnetic stirrer to redistrubute all the activated charcoal that has settled down.

I hope my explanation is clear and easy to understand. If there are any questions, please feel free to ask me. Thanks for taking the time to go through this post.


Johan
0606637G
TG01

Sunday, July 20, 2008

Week 4 SIP/MP Experience

Hello my fellow coursemates! How are you guys doing? Hope all of you are doing fine for your SIP and MP. This is the 4th week of SIP/MP and it is my pleasure to share my attachment experience.

I am attached to a research laboratory which study on the effect of polyphenolic chemosensitisers and anticancer drugs on tumour cells. Some examples of anticancer drugs include melphalan, chlorambucil and etc. Both chemotherapy drugs belong to the same class of nitrogen mustard alkylating agents. Melphalan is commonly used to treat multiple myeloma (cancer of plasma cells) and ovarian carcinoma. Chlorambucil is used primarily to treat chronic lymphocytic leukemia (cancer of lymphocytes, with particular to B cells). Some examples of polyphenolic chemosensitisers include 2,2'-dihydroxychalcone and 2'-hydroxy-4-methylchalcone (I shall elaborate more on polyphenolic chemosensitisers in my next post).

In the world today, there are 3 main types of cancer therapies which are chemotherapy, radiotherapy and surgery. Usually, they are used in combination to achieve greater effectiveness in treating tumours. My main focus will be on chemotherapy.

Chemotherapy is decreasing in its effectiveness mainly due to chemoresistance of tumour cells. This is primarily due to the gluthatione (GSH)-related detoxification system which allows tumour cells to be resistant to chemotherapy drugs. I shall elaborate further on this system in my next post.

In my study, I shall be focusing on the effect of 2,2'-dihydroxychalcone and chlorambucil in inducing DNA interstrand cross-links on human colon adenocarcinoma cells (HCACs) grown in-vitro. The HCACs are obtained from ATCC (American Type Culture Collection) and designated by COLO 320 HSR. These cells are grown in RPMI (Roswell Park Memorial Institute) medium and they are loosely adherent to the culture surface. In addition, they appear round and refractile under the inverted microscope. Since they are tumour cells, they divide very rapidly, and thus have high energy requirement.

During the course of my research, I am required to maintain these cells in peak condition so that I will have enough cells to continue my experiments. Hence, I will be applying what I have learnt from Mammalian Cell Technology (MCT) which I have taken as elective subject to part of my research. Without further delay, let me start off with the most fundamental, which is the preparation of the RPMI-1640 medium.

Subject: MCT
Tests:
1)Preparation of RPMI-1640 medium
2)Subculturing of cells
3)Changing of medium


1) Preparation of RPMI-1640 medium

Composition

· 1% sodium pyruvate
· 1% non-essential amino acids
· 1% antibiotics
· 10% fetal bovine serum (FBS)

Materials

· 500ml RPMI-1640 medium (ready-to-use)*
· Fetal Bovine Serum (FBS)*
· Antibiotics*
· Sodium pyruvate*
· Non-essential amino acids (NeAA)*
· BSC 2
· 70% ethanol
· Paper towels
· Pipettorˆ
· Falcon™ pipette tubes (5ml)ˆ

Methods

A. Preparation of BSC 2

1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.

B. Preparation of RPMI-1640 medium

1. Place the materials* in 37ºC waterbath.
2. Place the materials* into the BSC 2.
3. Pipette 5ml of sodium pyruvate into the pre-made 500ml RPMI-1640 medium.
4. Pipette 5ml of NeAA into the pre-made 500ml RPMI-1640 medium.
5. Pipette 5ml of antibiotics into the pre-made 500ml RPMI-1640 medium.
6. Pipette 50ml of FBS into the pre-made 500ml RPMI-1640 medium.
7. Store the medium at 2-8 ºC and warm up in 37ºC waterbath before any usage.

Function of components:

· Sodium pyruvate- Provide additional source of energy for rapidly-growing cells.
· NeAA- Provides source of amino acid (energy source for cells).
· Antibiotics- Minimises bacterial contamination of tissue culture.
· FBS- Provides essential nutrients to promote optimal cell growth and proliferation

2) Subculturing of cells

Principle

As cells grow and multiply, they tend to crowd together, which is termed as confluency. This can be observed under the inverted microscope where cells are in close proximity. A confluent tissue culture flask suggests the need for subculturing. Subculturing is defined as the inoculation of cells from a confluent flask into a new sterile flask with fresh medium. It allows cells to have more growth surface so as to minimize the competition for growth surface and nutrients. Subculturing is especially important for maintaining the viability, growth and proliferation of anchorage-dependent cells as these cells need to adhere to the surface of the tissue culture flask before they can start growing.

Materials

· 75cm² tissue culture flask (containing HCACs of 80% confluency)
· New 75cm² tissue culture flaskˆ
· RPMI-1640 medium*
· 0.0067M phosphate buffered saline (PBS)*
· Trypsin*
· Inverted light microscope
· BSC 2
· CO2 incubator
· Pipettorˆ
· Sterile 50ml Falcon™ tubeˆ
· Falcon™ pipette tubes (25ml and 5ml)ˆ
· Waste beakerˆ
· Biohazard bag
· Clorox (Bleach)
· 70% ethanol
· Paper towels

Methods

A. Preparation of BSC 2

1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.

B. Preparation of reagents

1. Incubate trypsin and RPMI-1640 medium in 37ºC water bath.

C. Washing of HCACs

1. Remove the 75cm² tissue culture flask (containing HCACs of 80% confluency) from the CO2 incubator.
2. Observe cell confluency (80%) under inverted light microscope.
3. Swab the 75cm² tissue culture flask with 70% ethanol before placing it into the BSC 2.
4. Swab the materials* before placing them into the BSC 2.
5. Discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker.
6. Pipette 10ml of 0.0067M PBS into the 75cm² tissue culture flask to wash the HCACs.
7. Swirl the 75cm² tissue culture flask gently to facilitate the washing of HCACs.
8. Discard 10ml of PBS into the waste beaker.

D. Trypsinisation

9. Pipette 2ml of trypsin into the 75cm² tissue culture flask to detach the HCACs from the tissue culture flask surface.
10. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2 for 2-3 minutes.
11. Observe the 75cm² tissue culture flask under the inverted light microscope to ensure the HCACs are detached.

E. Subculturing of cells

12. Pipette 8ml of fresh RPMI-1640 medium into the 75cm² tissue culture flask to neutralise trypsin to prevent damage to the HCACs.
13. Mix the RPMI-1640 medium with trypsin well to ensure all the HCACs can be transferred with the RPMI-1640 medium into a sterile 50ml Falcon tube.
14. Pipette 10ml of mixture (8ml of RPMI-1640 medium and 2ml of trypsin) from the 75cm² tissue culture flask into a sterile 50ml Falcon tube.
15. Centrifuge the sterile 50ml Falcon tube at 20ºC, 1500rpm for 3 minutes.
16. Discard the supernatant into the waste beaker, leaving the cell pellet.
17. Resuspend cell pellet in 10ml of PBS.
18. Centrifuge the sterile 50ml Falcon tube at 20ºC, 1500rpm for 3 minutes.
19. Discard the supernatant into the waste beaker, leaving the cell pellet.
20. Resuspend cell pellet in 10ml of RPMI-1640 medium.
21. Pipette 19ml of fresh RPMI-1640 medium into a new 75cm² tissue culture flask.
22. Pipette 1ml of cell suspension from the sterile 50ml Falcon tube into a new 75cm² tissue culture flask.
23. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2.

F. End of experiment

1. Deactivate the waste solution in the waste beaker with Clorox.
2. Drain the waste solution into the sink after it turns from violet to colourless and run running tap water for 5-15 minutes.
3. Dispose all the used Falcon™ pipette tubes and paper towels and into the biohazard bag.
4. Swab the BSC 2 with 70% ethanol.
5. Return all the reagents to their appropriate storage areas.

3) Changing of medium

Principle

As cells grow and multiply in culture medium, they produce metabolic toxic waste products and use up nutrients in the medium. Essentially, we want to maintain the viability of cells and thus, the changing of medium is necessary as it restores back the level of nutrients and removes metabolic toxic waste products. An indication of the need to change medium is by observing the colour of the medium. Phenol red is added in the medium to act as a pH indicator. A yellow culture medium indicates the accumulation of metabolic toxic waste products and depletion of nutrients, prompting the need to change medium. A cloudy culture medium suggests possible bacterial contamination and the best action would be to discard the tissue culture flask.

Materials

· 75cm² tissue culture flask (containing yellow culture medium and cells)
· 0.0067M phosphate buffered saline (PBS)*
· RPMI-1640 medium*
· BSC 2
· CO2 incubator
· Pipettorˆ
· Falcon™ pipette tubes (10ml and 25ml)ˆ
· Waste beakerˆ
· Biohazard bag
· Clorox (Bleach)
· 70% ethanol
· Paper towels

Methods

A. Preparation of BSC 2

1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.

B. Changing of medium

1. Remove the 75cm² tissue culture flask (containing HCACs of 80% confluency) from the CO2 incubator.
2. Swab the 75cm² tissue culture flask with 70% ethanol before placing it into the BSC 2.
3. Swab the materials* before placing them into the BSC 2.
4. Discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker.
5. Pipette 10ml of 0.0067M PBS into the 75cm² tissue culture flask to wash cells.
6. Swirl the 75cm² tissue culture flask gently to facilitate the washing of cells.
7. Discard 10ml of PBS into the waste beaker.
8. Pipette 20ml of fresh RPMI-1640 medium into the 75cm² tissue culture flask.
9. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2 for 2-3 minutes.

C. End of experiment

1. Deactivate the waste solution in the waste beaker with Clorox.
2. Drain the waste solution after it turns from violet to colourless.
3. Dispose all the used Falcon™ pipette tubes and paper towels and into the biohazard bag.
4. Swab the BSC 2 with 70% ethanol.
5. Return all the reagents to their appropriate storage areas.

In conclusion, subculturing of cells and replacement of medium is essential for maintaining the viability of cells. Alright, that is all for now! I hope my post is comprehensive and if there is any query, please feel free to ask.

Thankz! =)

Tan Han Yang
0606190G
TG01

Saturday, July 12, 2008

Week 3 SIP - Pilot Food Catering Tech Plant and my beloved MP

Heya guys, hope you all have been enjoying your SIP because i have. I have been attached to the Pilot Food Catering Plant and have started my SIP this week. I am sorry i cannot share any recipes with you guys because it is a trade secret .

There are many jobs to do in a food technology plant. You will have to help the chefs prepare ingredients, perform baking of pastries, grinding of cashew nuts, labeling of food containers, sieving of flour, cracking off eggs, packaging of cakes, maintenance of machines, cleaning the facilities and greasing the baking trays. And this is only the 1st week of my attachment there!!

The preparation of ingredients is very important in baking a premium pastry or cake. Hence it is very imporant to get your measurement units right. A kilogram does not equate to a gram of sugar. Imagine adding a kilogram of sugar into your pastry... YUmYUm.

After the baking proccess and the finished product is achieved(hopefully it turns out well), the TSO and i will have a food tasting session. We will sample the quality of the food and a checklist will be evalutaed. The checklist consist of gradings from 1 to 5 and is very important to gauge the quality of the food before it can be moved to Bristol to be sold. These checklists will not be only given to us, but also to the rest of the students from the Food science and culinery courses for their remarks. Basically the checklists will ask questions on the texture (soft, hard), taste (sour, bitter, sweet, sour), colour (pale, dark, brown), enjoyment (enjoyed eating, feel like puking) and further remarks on how i can be improved. All the checklists wil be gathered and keyed into the computer (not LIS) and tabulated using Excel to find the average response to the food. If the average pertains to liking the product (majority), the cakes/pastrieswill be packaged and labeled before shipped across to the Bristol to be sold.

My Mp involes developing a protocl for zymogram and i briefly explain the principles of a zymogram.

Zymography is a type of electrophoresis technique using SDS-PAGE. It is different from the usual electrophoresis, as it is copolymerised with a substrate (usually geltain or caesin) Why this 2 substrates? Most proteases and periplamic proteins are able to digest there 2 substrates and this allows us to measure and detect the enzyme activity. Another difference for zymogram and normal electrophoresis is that the sample buffer is prepared without boiling and adding of reducing agent such as B-mecapethanol. This is because we do not want to denature the protease and periplasmic proteins as we want them to breakdown the substrate to detct the enzyme activity. After electrophoresis, Triton X-100 is added for the renaturation process and incubated in the digestio buffer for the reaction to take place.Later the zymogram is stained with coomassie blue stain and will show up as clearings/halos whereby there is digestion of periplasmic proteins to the substrate have taken place.

Thanks for reading my entry, have a great MP/SIP ahead

From: Benjamin MA
Class: TG01
0606181F
Date: 12/07/08

Saturday, July 5, 2008

Week 2 SIP - Xcise Machine

Name of topic: Xcise

Introduction to the Topic: My MP involves the analysis of Stenotrophomonas maltophilia’s periplasmic proteins. Periplasmic proteins are proteins that are found between the the inner and outer cell wall. The study of these proteins are important as it could be potential drug targets in the future. During the course of this study, quite a number of machines would be needed.

The machine im going to blog about in this entry is the Xcise machine

So what is Xcise? Xcise is an automated gel processor that is useful in processing proteins that are to be identified by mass spectrometry. Its functions ranges from acquisition of gel image to the spotting of protein sample onto a MALDI target plate. Before we can use Xcise, 2-D gel electrophoresis should be performed 1st.

2D gel electrophoresis: Proteins are separated twice during gel electrophoresis. For the 1st dimension, proteins are separated using an IPG (Immobilized pH Gradient) strip according to their isoelectric point. The proteins move horizontally. For the 2nd dimension, proteins are separated using a pre-casted gel based on their molecular weight. This time, proteins move vertically downwards.

Isoelectric point: Isoelectric point is a characteristic of the protein whereby it corresponds to the pH at which the protein is neutrally charged. This means that the proteins will migrate on the IPG until it reaches the pH of the strip, whereby the protein is neutrally charged.

Mass Spectrometry: A technique used to identify and sequence proteins by measuring the mass-to-charge ratio of proteins that are converted to ions. The instrument used to measure the mass spectrum is called MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight).

Matrix compound: A compound that is required to control the energetics of the desorption/ionization process.

After a gel is run, proteins are already separated based on their isoelectric point and molecular weight (2D gel). Proteins separated would appear as spots on the gel. The protein spots would then be stained for visualization and a gel image is acquired. After the gel is stained, we would want to identify the proteins. Before running the proteins through MALDI, the proteins would need to be removed from the gel, digested into peptides and then spotted onto a MALDI target plate.

Outline of Xcise’s in-gel digestion procedure


1. Gel image is acquired and spots to be cut are selected
2. A cutting head will cut out the gel containing the protein spots and place them into wells
3. The gel will then be destained and dehydrated
4. Trypsin is added to digest the proteins into peptides (note that the proteins are still within the gel)
5. Sample is then incubated for 4-6 hrs at 37oC or 16-18 hrs at 30oC

6. As buffer used contain salts that will affect the subsequent analysis of peptides in MALDI, peptides would need to be desalted
7. Peptides are then eluted using ZipTip. ZipTip is different from a normal pipette tip as it contains a resin at the tip. Peptides would bind to the resin during the process of desalting
8. The peptides are then spotted onto a MALDI plate together with a matrix compound
9. The MALDI plate can then be placed in MALDI and peptides can be analysed


LMQA (How can excise be helpful?)

Using Xcise is an example of lab automation. If the process is to be done manually, it will be very tedious and time consuming. For the cutting of gel, the lab technician would need to measure diameter of the protein spot, before cutting a pipette tip so that the hole corresponds to the diameter. The pipette tip will then be used to cut the gel. During cutting, the gel would be lodged inside the tip and would have to be taken out using a pointed end. This makes cutting extremely difficult. Steps 3-7 would need to be done manually too by adding and removing the required solutions manually.

For the spotting onto MALDI plate, lab technician would have to spot onto the plate one by one. One spot will be placed in one eppendorf tube so if there are 100 spots, there will be 100 tubes to be processed and 100 spots to be spotted onto the plate. This may lead to fatigue and air bubbles in the sample may be produced. However, if Xcise is used, the cutting of gel would be faster and more precise, hence decreasing the amount of varaitions. At the spotting stage, Xcise can spot 8 samples at a time, which is much faster and accurate than doing it manually. Contamination of the sample is also greatly minimized. Even though the machine and the consumables are very expensive (1 ZipTip costs about $2, and 8 ZipTips will be used at a time), it can greatly improve the efficiency of the lab due to its importance in maintaining the integrity of the sample.


Posted by: Ma Xianwei Benjamin

Class: TG01

10.15am

05/July 2008

Friday, July 4, 2008

Week 2 SIP (Ivan)

I am posted to the biochemistry/clinical chemistry section of my lab for 2 weeks. For this 2 weeks, I have been doing 2 kinds of major test on EDTA-coagulated blood specimens. One is G6PD and the other is HbA1c Quantitation. I will be putting up a post regarding HbA1c Quantitation.

At the end of my first week, I have learnt a new constituent in the blood, which can be tested. It is HbA1c or Glycosylated (or glycated) hemoglobin. Glucose in the blood stream will normally be attracted to the haemoglobin part at the lysine molecule of the RBC to form glcosylated haemoglobin. Thus the amount of HbA1c is directly proportional to amount of glucose, meaning that if there are more glucose present in the blood, the more HbA1c will be present in the blood. It’s a known fact htat RBC has a half-life of about 3 months before they are replaced by the spleen. Hence by measuring the percentage of HbA1c in the blood, we can determine how high the patient’s blood glucose has been on average over the last 3 months.

Currently HbA1c is the best suitable way to monitor the progress of medication for patients suffering from diabetes mellitus. It is also able to determine the appropriate dosage quantity of anti-diabetic drugs do administer to patients so as to effectively reduce glucose blood level and reduce possible side-effects.

I am now working in a company who uses a machine manufactured in Japan to quantitate in percentage the amount of HbA1c in the blood specimen. Me as a lab technician,I am required to load the blood specimen manually. However before loading the blood specimen, I am instructed to shake and mix well every blood specimen and it is important to remove any air bubbles present using a disposalable plastic pipette. This is becase the machine has a laser to detect the level of blood in the tube,it will not go all the way to the bottom to aspirate blood. Thus presence of air bubbles will give false level of blood detection, causing inaccurate amount of blood aspirated, in the end causing false results.

The principle of my company’s machines is as follows; the concentration of HbA1c and the concentration of total haemoglobin in the blood sample are measured separately, and then the ratio is reported as percentage of HbA1c. A latex agglutination inhibition method is used for measurement of specific HbA1c. The total haemoglobin is then measured using the toral haemoglobin reagent where all derivatives are converted into alkaline hematin in an alkaline solution of a non-ionic detergent.


By Ivan Ng
TG01
0605070B
11.40pm
4 July 2008 (Friday)