Lab Techniques

Trichloroacetic acid(TCA) is one of the many organic solvents used in protein precipitation and purification. It works by disrupting the protein conformation thus exposing more hydrophobic areas, which reduces solubility of protein. In this way, the proteins will be precipitated.

We are using TCA to extract secretory proteins from our bacteria (S.maltophilia) and these are the steps involved.

Day 1 (Streaking)
1. Streak the strain on a LB Agar plate
2. Incubate plate for 24 hours at 37°C

Day 2 (1st Inoculation)
1. Inoculate an isolated colony into 20ml of LB broth
2. Incubate tube for 24 hours at 28°C
(Remark: The tube is incubated at 28°C instead of 37°C as most of the strains grow better at about 30°C. We'll like to the bacteria to proliferate so there will be enough cells for 2nd inoculation and for extraction)

Day 3 (2nd Inoculation)
1. Pellet cells at 3000xg for 20 minutes at 10°C
2. Decant the supernatant and resuspend the cells in 10mL of PBS

3. Spin down the tubes at 3000xg for 20 minutes at 10°C

4. Repeat steps 2 and 3.

5. Decant the supernatant and resuspend the cells in 20mL of PBS

6. Take OD600 reading in triplicates with 10X dilution
7. Calculate the volume withdrawal equivalent to 5.0X107 cells
8. Inoculate the calculated volume each into 4 tubes of 20ml LB broth
9. Incubate tubes at 37°C for 16 hours

Day 4 (TCA Precipitation)
1. Pellet cells at 3000xg for 20 minutes at 10°C
2. Suck up the supernatant (which contains the secretory proteins) with a syringe
3. Filter 18 ml of supernatant through 0.45um filter to remove contaminants (e.g. cell debris) into teflon tubes.
4. Add equal volume of chilled TCA into each tubes
5. Incubate the tubes -20°C freezer for an hour
6. Invert tubes every 10 minutes interval
7. Spin tubes at 16 000xg, 4°C for an hour

8. Decant the supernatant (beware of dislogded protein pellet)

9. Wash the protein pellets with 250ml of pre-chilled acetone
10. Transfer all protein pellets into a 1.5ml mircofuge

11. Centrifuge at 16 000xg at 4°C for an hour

12. Aspirate supernatant and air dry

13. Resuspend pellet with appropriate amount of rehydration buffer

Acetone is used to wash the cell pellet as TCA is also an organic solvent, like dissolves like so the TCA can be removed. Also,chilled acetone is preferred as low temperature allows the protein to stay in ppt form. When room temp acetone is used, protein pellet turns sticky and thus stick onto pipette tip and tube leading to loss of protein sample. Furthermore, the addition of organic solvent causes heat evolution, care should be taken to keep the temperature low.

Afterwhich, we'll do a protein quantitation to determine the concentration and run a 2D electrophoresis (refer to my first entry), followed by analysis of spots and excision of chosen spots(refer to Ming boon's entry). Finally, to run these peptides in MALDI TOF/TOF (refer to Shahirah's entry) to identfy these peptides. Scroll down if you want to read more about the principles of using these equipments.

That's all! It's the last week that we needa blog! YEAH. Hope you understand and learn something from my posting. See you people in school!

Tang Jiaxin (TG01)

Lab Techniques

Hi guys...today i'll be talking about the washing procedures that Zahirah and I always have to do when there is insufficient sequencing plates and Axymats for our experiments. Hee... Feel free to ask any questions ya...

Title: Cleaning of sequencing plates procedure

1. Flood labeling on the sequencing plates with DMSO and wiping the sequencing plates dry and clean with tissue papers.
2. Rinse sequencing plates with tap H₂O.
3. Add decon 90into sonicator.
4. Place all sequencing plates into sonicator. Ensure they are totally submerged by using Styrofoam to press them down and the close the lid of the sonicator. Then add distilled H₂0 into the sonicator, filling it ¾ full.
5. Turn on the sonicator for 30min. (It will induce electrical impulses for washing of sequencing plates.)
6. After washing for 30min, turn on the tap of sonicator to allow the washing solution to be drained out into an empty pail and later been disposed into the sink.
7. Add decon 90 into sonicator again.
8. Rinse sequencing plates with tap H₂0 again.
9. Place sequencing plates into sonicator and add distilled H₂0,filling it ¾ full again. Place Styrofoam to press down the sequencing plates to ensure that they are fully submerged into the washing solution.
10. Turn on sonicator for 15 min.
11. After 15 min, turn on the tap of the sonicator to drain off the washing solution.
12. Rinse sequencing plates with tap H₂O again.
13. Take a beaker to fill up with milliQ H₂O.
14. Rinse the sequencing plates in milliQ H₂O for 4-5times.
15. Dry them using tissue papers.
16. Place them in the oven(65^oC) for 1 hour.
17. Wrap them with aluminium foil and sent them for autoclave.

Title: Cleaning of Axymats procedure

1. Prepare 0.5% to 1% sodium hypochlorite (bleach) solution. In this case, 1% bleach is prepared using M₁V₁=M₂V₂.

5.25% of bleach (stock conc) x V₁= 1% of bleach x 1500ml (total volume of bleach solution required)

Thus, V₁= 286ml (approx. 300ml)

Hence, add 300ml of 5.25% bleach into 1200ml of milliQ H₂0 and pour the solution into the sonicator. Turn on sonicator for 15 min after placing the Axymats into the prepared 1% bleach solution.

2. Rinse Axymats into 70% isopropanol and allow them to dry on tissue paper.
3. Place them in the oven for 30 min.
4. Wrap them with aluminium foil and sent for autoclave.

Michelle (0503808h)
TG02

SIP/MP sharing week 16

Hey there! Yup it's my turn once again. Time really flies. It's week 16!!!

In this entry, i will be sharing with you my SIP/MP experience. As mentioned by both Jiaxin and Ming Boon, we have been assigned to be 'supervisors' to the DRP students for about a month. Ming Boon and myself have been tasked to supervise the students doing the protease project (refer to Ming Boon's post on 30 Sep). However, the students from the other project will also be supervised by us should both Johanna and Jiaxin be busy. As much as it was interesting, I must say I was thrown off guard even though I was fully aware of the fact that they had never worked in this conditions before. Frankly, I think I know how the lecturers feel now :)

I'd like to share with you guys an incident that happened recently. The group working on the extraction of periplasmic proteins using chlorofrom shock (refer to Jiaxin's post on 17 Sep) were supposed to run their sample on a 1-D gel (think MBio, Western Blot). They had prepared their samples as per protocol (or so i thought). After running their samples on the gel, it strucked them that they did not denature their samples.

For the benefit of everyone, I shall sidetrack and explain the flow for running a pre-casted 1-D gel.

Basically, you will take out a certain volume of the samples into a new microcentrifuge/eppendorf tube. In this case, it was 10ul. After which, you will add an equal volume of a mix of sample buffer (I'm not sure what's in it but it contains Bromophenol Blue and SDS [gives the proteins an overall negative charge]) and B-mercaptoethanol. Any idea what's B-mercaptoethanol is for? It is a strong reducing agent that breaks the disulfide bonds found in proteins. (This step is done under the fume hood as B-mercaptoethanol gives off a very pungent smell.)


This image was taken from http://tonga.usip.edu/gmoyna/biochem341/bme.gif

Once you have done all that, you will centrifuge the tubes for 4-6 secs to collect all the liquid down. Then you will heat the samples up at 95°C for 5 mins to denature the proteins. Following this, the tubes will be centrifuged briefly again to collect all the liquid down. The samples are now ready to be loaded onto the gel.

The gel is removed from its packaging (the gel is pre-casted) and placed in the gel tank. All the samples are loaded in the wells, except for the first and the last wells where the protein ladder/marker will be added. You will then top up the buffer in the gel tank if needed and the gel is noe ready for electrophoresis. Yup so that's the steps done when running a 1-D gel. For those who are interested to know what happens after that, leave me a comment aite?

Back to the actual story. As I'd said earlier, they did not denature their samples . This means that the proteins will still be in their native conformation and thus affect their rate of migration in the gel, which in a way is pointless as the main principle of 1-D gel is to separate the linearized proteins based on their mass.

This will be one incident that I will not forget.

That wil be all for this week's post. To all, Selamat Hari Raya and hope you will have a good weekend. Four more weeks to go!!!! Take care and see you guys during the next campus discussion!

Posted by Shahirah Bibi Tg01, 0503174E

MMIC

Procedure: Biochemical Testing for Neisseria gonorrhoeae

Posted by: Azhar Hamdan (0503269C)

For the moment, I am temporarily attached to the Serology Lab for 2 weeks, with my first week being in the STD Lab. Yeap, thats rite Sexually Transmitted Diseases. I know what you are thinking, "Eeeks HIV!" However, on the contrary , the lab only tests for 2 organisms, Neisseria gonorrhoeae (causes Gonorrhoeae) and Treponema Pallidum (Syphilis). So no HIV here :).
For this post, I am going to try adding as much pictures as possible to keep you guys interested alrite?

Anyways my post will focus more on N. gonorrhoeae, a.k.a Gonococci or GC.

The common types of specimens received by the lab are swabs from:

• Endocervix
• Urethra
• Anorectal
• Oropharynx
• Conjunctiva
• Bartholin gland
• Skin lesions
• Joint fluids

And plain blood specimens.

An interesting point to note is that more than half of the specimens come from Sex Workers that regularly go for checkups to retain their license. So people, always stay protected!

Anyways, upon receiving the specimens, the lab staff would process the specimens and allocate lab numbers. They would then inoculate & streak the swabs onto GC -Lect Agar, a selective agar that promotes the growth of GC that contains antibiotics that would suppress the growth of other organisms such as gram-positive bacteria, including vancomycin-resistant Staphylococcus epidermidis, gram-negative species like Proteus and Capnocytophaga, as well as fungi including Candida albicans. The plates would then be incubated at 35-37°C for 24 hours.

All the above is done by the lab staff.

After incubation, the plates are read. Suspected GC colonies undergo a Gram-stain & 4 biochemical tests (these were practised by us students):

• Oxidase test to test for the production of oxidase
• Superoxol test (using 30% Hydrogen Peroxide [H₂O₂]), similar to the Catalase test (using 3% H₂O₂), to test for the production of catalase
• Rapid Carbohydrate Degradation Test (Sugar Test), to test for the ability to degrade sugars)
• β-Lactamase test, to test for the production of penicillinase which confers resistance to penicillin

Only the first three are confirmatory tests. The last test is a susceptibility test to check for resistance to penicillin.

The oxidase test and superoxol test (similar to catalase) are some common tests that we have learnt in Basic Microbiology so I shall not go into details, however, questions are welcomed.

I chose the Rapid Carbohydrate Degradation Test (Sugar Test) to elaborate on, as it is so different from all the other tests that checks for the utilisation of sugar (e.g. TSI & KIA). Specimens are tested with the sugars individually. The sugars used in this test are 10% solutions of Glucose (G), Maltose (M), Lactose (L) and Sucrose (S)

Rapid Carbohydrate Degradation Test (Sugar Test)

Principle
This is a non-growth method which depends upon pre-formed enzymes. A heavy inoculum of organism degrades the sugar solutions containing the phenol red indicator. A colour change occurs in 2 to 4 hours.

Procedures

1. A heavy suspension of the organism is made (using a platinum inoculating loop) in a test tube of 1.0mL Buffered Salt Solution (BSS), to contain approximately 10⁹ organisms per mL. Controls were also done together with the patient specimen. These controls are N. gonorrhoeae CDC 117 strain (117), N. lactamica (LC), Branhamella catarrhalis (BC) & Staphylococcus epidermidis (S)The BSS is red in colour due to the phenol red indicator.
2. This suspension is mixed well with a Pasteur pipette (Two Pasteur pipettes are available, use the bigger pipette [top] for this step).
3. Using the bigger Pasteur pipette, two drops of the suspension are delivered into each of the five wells of a microtitre plate which are labeled horizontally across the plate as Control (C), Glucose (G), Maltose (M), Lactose (L) & Sucrose (S).[The group of wells on the left side were from a previous practice, microtitre plates are shared to avoid wastage]
4. Using the smaller Pasteur pipette, one drop of each of the 10% carbohydrate solutions (G, M, L and S) is added carefully to each of the appropriate wells, making sure there is no splashing.
5. Cover the microtitre plate and mix well using the micro-shaker.
6. Incubate at 35-37°C for 2-4hrs
7. After incubation, examine plate for colour change. A red to yellow colour change indicates a positive reaction. Control well should remain an orange/red colour which indicates a negative reaction.
   Results for the above test (both left & right specimens):
   • First row, patient specimen: positive for G.(N. gonorrhoeae)
   • Second row, 117: positive for G.
   • Third row, LC: positive for G, M & L.
   • Fourth row, S: positive for G,M & S
   • Fifth row, BC: all negative

All the results tally with the expected results.

Pictures were taken with permission from the supervisor. Thanks Andre for being my model. Haha.

So thats all.

5 more weeks...Ahhhh!!!

Lab Techniques

Hi everyone,

Like Jiaxin and Johanna, I am also the ‘supervisor’ of the DRP students. Shahirah and me are taking the group that will be doing a project that involves proteases secreted by Stenotrophomonas maltophilia at 37^oC. We would have to teach them how to prepare the necessary media, teach them the things that they do not know (eg. how to streak in the proper way) and guide them through the experiment so that it is successful.

This is a very simple project and the purpose is to find out which are the strains that produce a lot of proteases. Strains that produce a lot of proteases might be more virulent as the proteases might be involved in the degradation of the host proteins involved in the immunity eg. Immunoglobulins (Ig).

Principle:

After growing the colonies, the colonies will be transferred onto milk agar. If the strain produces proteases, the proteases will digest the milk proteins and a clearing that looks like a halo around the colony will be observed.

Procedure:

Day 1 - Streaking of environmental strains

1. Streak the strains onto LB agar (Plate is divided into 2 as there are only 2 strains and only abit of the colony is needed)
   
   
2. Incubate the plate at 37^oC for 48 hours (As environmental strains do not grow very well at 37^oC, more time is required for them to grow)

Day 2 - Streaking of clinical strains, negative and positive control

1. Streak Escherichia coli (negative) and Pseudomonas fluorescens (positive) onto a LB agar each
   
   
2. Streak the strains onto LB agar (Plate is divided into 4)
   
   
3. Incubate at 37^oC for 24 hours

Day 3 - Transferring of colonies to milk agar

1. Divide and label the milk agar appropriately
   
   
   
2. Use a sterilised toothpick to touch the colony and then dab on the milk agar gently
   
   
3. Incubate the plates at 37^oC
   
   
4. Observe for halos (This will take about 3 days of incubation)

That’s all! Thanks=]

Chua Ming Boon

Tg01

0503197F

Florescence in situ hybridization

Florescence in situ hybridization (FISH), is a technique involving the separation of double stranded DNA by heat and hybridization of the target DNA sequence to the respective probes with a complementary sequence deirectly-labeled with a florochrome. The metaphases to which the probes anneal to are visualized as florescent signals of specific wavelengths under fluorescence microscopy. The number of signals observed in the metaphase spreads/interphase cells would indicate the copy number of the gene/gene loci concerned. This technique allows the identification of microdeleted/duplicated genes, aneuploidy screening (detection of extra chromosomes of chromosomes 13, 18, 21 X and Y), translocated genes in haematological malignancies and gene amplification from cases with cancer of the breast.

I would be explaining on identifying deleted/duplicated genes and loci that are related with microdeletion syndromes in congential disorders such as Prader-Willi/Angelman, Williams and Di-George using FISH techniques.

Samples – cultures of PHA(mitogen)-stimulated lymphocytes cells, amniotic fluids and chorionic villi.

Slide Preparation

1. Etch out the area on the underside of the glass slide with a diamond pen that contains the highest number of metaphase spreads. Slides can be kept in an airtight container at -20°C or 4°C if they are not used immediately.

2. Dehydrate the slide through an ethanol series (70%, 85% and 100% respectively) for 1 minutes each at room temperature. Blow-dry the slide with a hair dryer.

Probe Preparation and hybridization
The following steps must be carried out in reduced lighting as the probes are light-sensitive.

1. Remove the probe vial from the freezer and thaw at room temperature. Centrifuge the vial.

2. Add probe mixture onto the etched area of the slide and cover with a circular glass coverslip.

3. Seal the edges with rubber cement and place the slide into a light proof box.

4. Switch on the 'HYBrite' system and set the desired program (denaturation and hybridization steps).

5. Moisten the toweling paper in the troughs with water. Place the slide onto the hybridization surface, coverslip facing upwards.

6. Ensure that the target area is in contact with the hybridization surfaces and also that all the remaining slide positions are filled with blank slides to even out the temperature.

7. Close the 'HYBrite' lid and commence the program.

Post Hybridization Wash
Carry out the following steps in reduced lighting.

1. Switch on water bath. Set the temperature to 75° C. Prepare two coplin jars for Wash A and Wash B or two plastic slide chambers if only 1 or 2 slides are required for washing.

2. Place the coplin jar/slide chamber containing Wash A in to the water bath when it is just switched on.

3. Leave the coplin jar/slide chamber containing Wash B at room temperature.

4. When the water bath reaches its set temperature, remove the slide from the 'HYBrite' system. Peel off the rubber cement from the coverslip with a pair of forceps. Remove the coverslip.

5. Place the slide in the Wash A and agitate the slide by jiggling briefly for about 3 seconds. Wash for exactly 2 minutes.

6. Remove the slide and transfer to Wash B. Agitate the slide by jiggling briefly for about 3 minutes. Wash for 1 minute.

7. Remove the slide and air or blow dry completely in the dark at room temperature. Apply 10m l of DAPI counterstain (Vectashield mounting medium) and mount a glass coverslip over the hybridize area. Seal with nail vanish.

Image of Digeorge Syndrome

Adapted from :
http://www.pathology.washington.edu/galleries/Cytogallery/main.php?file=intro

Image of HYBrite system

Adapted from:

http://www.ebiotrade.com/buyf/productsf/vysis/img189_1.jpg

Reference:

American College of Medical Genetics Laboratory (1999). Standard guidelines for clinical genetics laboratories, 2nd Ed. Bethesda, MD: ACMG.

By:

Yvonne Lau

0503149G

TG01

Lab Techniques

HELLO! =D

If you're wondering what we the in-house students are doing for SIP. NO NO.. not preparing reagents and stock checks! For our group, our assigned task is to be the "supervisors" of the DRP (Differential Research Programs) students!

Johanna and I will be taking one of two groups who are under Dr. Quek's project. This project involves extraction of periplasmic proteins using chloroform (CHCl₃) shock, of an isolate of Stenotrophomonas malitophilia which is incubated at two different temperature 28°C (environmental) and 37°C(human body temp.). The differences in protein expression will be analyzed and compared. Experiment will be repeated on other isolates too. This is also the continuation of what Jo and I have been doing for our last DRP.

Outline of Workflow/Protocol

Day 1 (Streaking)

1. Streak the different isolates on a well divided LB Agar plate
2. Incuabte plates for 24 hours at 37°C

Day 2 (Inoculation)

1. All colonies of each isolates is scrapped off and mix in 5mL PBS
2. Cell density (Opitcal Density 600) taken in triplicates to determine number of cell present
3. Volume of inoculum equivalent to 5.0X107 is inoculated into 2 tubes
4. One tube incubate at 28°C and the other at 37°C for 16 hours 250 rpm

Day 3 (Extraction)

1. After 16 hours, the tubes are spin down at 3000xg for 20 minutes at 10°C

2. Decant the supernatant and resuspend the cells in 10mL of PBS

3. Spin down the tubes at 3000xg for 20 minutes at 10°C

4. Repeat steps 2 and 3.

5. Decant the supernatant and resuspend the cells in 10mL of PBS

6. Take OD600 reading in triplicates with 10X dilution

7. Calculate the volume withdrawal equivalent to 1010 number of cells

8. Pipette the calculated volume into 1 microfuge tube

9. Spin down cells at 3000xg for 20 minutes at 10°C

10. Decant supernatant

11. Resuspend pellet in remaining medium by brief vortexing

12. Add 20mL of chloroform to each tube

13. Incubate at room temperature for 15 mintues

14. Add 200uL of 0.1M Tris-HCl to tubes to stop reaction

15. Spin tubes at 6000xg for 20 minutes at 10°C

16. Collect the supernatant to a new microfuge tube

17. Spin the supernatant tubes at 14000xg for 10 minutes at 10°C

- This is to ensure that only the periplasmic proteins are present in the supernatant as cell debris will be pellet down

18. The supernatant is pipetted out into a new microfuge tube

Example of how to calculate 5.0X107 worth of cells

Average OD600 Reading = 1.70

Desired number of cells = 5.0X107

1.00 OD = 1.4X107 cfu/mL

Volume of inoculum = (Desired no. of cell) / (Average OD600 X 1.4X107) X1000

= (5.0X107) / (1.70 X 1.4x107)

= 21.0uL

After extraction, protein concentration will be determined by Bradford assay. The samples will be run on 1-D gel (separation by MW) and stained with either Coomassive Blue or Silver stain. Protein bands will be analyse and compare to identify the difference in protein expression of an isolate incubated at 28°C and 37°C.

Post Stained 1D Gel
Adapted from: http://www.raytest.de/bio_imaging/applications/gel_documentation/applications_geldoc1.jpg

As this is a project that lasts for only 3 weeks, the job load is lesser and simpler.

That's all. Have great day!

Tang Jiaxin TGo1

0503257H