Lungs burnt!

After a long cold winter season, I went for a run this evening, thinking it was pleasant. I was in my tees and shorts only. While waiting for Vladimir to join me for the run, I felt the cold air (around 15 degree Celsius). I made a mental note that it would be alright after I started running.

Initially, the cold air felt refreshing. But after a short distance, the cold air just stunk my oxygen-deprived lungs and my skin pores contracted against the cold wind.

Next time, I would run on the treadmill or wait till the temperature climbs further up.

30 chocolate bars for snack

Yes, you got the number right, 30! I bought them as a variety pack consisting of 10 Snickers, 8 Twix (I love this so far), 3 Snickers Almond, 6 Milky Way and 3 Musteteers at Sam's Club where it sells items at wholesale quantity and price. The down side is that it does not carry much varieties.

Friends who had been to Europe and USA had told me that the chocolate and pastry taste different from that in Singapore. So, I am trying them out now. After finishing this pack, next on the list will be Hershey's brand of chocolate.

Too bad that Sam's Club stop carrying Goldiva chocolate ice cream, else I would be indulging in them. I am not really a fan of Häagen-Dazs. By the way, I am still stuck with two tubs of Edy's ice cream. Think they will stay in the freezer for a long time.

Home cooked dinner when I reached back

For the whole of this week, I get free home cooked dinner after the end of a hard day. Where do I get such a good deal, you would ask me?

My landlord's parents are here for a visit. His mum is doing all the cooking and I have a share of it. Nice not to be cooking your own dinner for a while. Besides, she cooks Chinese dishes such as steamed fish (which I have not eaten since I left Singapore), lotus root soup (detoxifying and 'cooling' for the body) and stir-fried leafy vegetables, etc.

Enjoying the bliss now.

Possibility of commuting between USA and Singapore

My boss, Prof. Thomas August is applying for a grant in Singapore. If he got it approved, I might be selected among his post-docs to travel to and fro between Baltimore and Singapore.

Of course I am hoping that the grant be approved and that I am chosen since I can see my family and friends more often in a year.

The down side is the 27hr-flight and adjusting to 12-hour difference. A small price to pay. For now, let me day dream till the verdict is out.

Ligation - Perserverance strikes back

After one month of ligation experiments, two out of three constructs were successfully subcloned into our lab's vector. For the last construct, I am using Vladimir's clone to save time.

This period had been frustrating and trying where after repeating so many experiments, the inserts were not ligated to the vector. I had tried different T4 DNA ligases from Roche, Promega and New England Biolabs. The ligation reaction mixtures had been incubated at room temperature, 16 degree celsius and 4 degree celsius. Incubation time tried were as short as 5 minutes to overnight.

Nearing the end of one month, I was feeling down and self-doubt set in. Many thanks to my friend, TuanLeng who encouraged me and discussed with me the possible mistakes in ligation. I perservered and for the last ligation experiment, I finally determined the vector and insert concentrations and calculated the volumes to react for giving a molar ratio of 1 (vector) : 3 (insert). Background and negative controls were set up. The background control was the vector without any insert while the negative control had insert only without the vector. Colonies from the background control helped to gauge the ligation efficiency. No growth should be observed on the negative control plate.

Lo and behold, when I saw the gel, I was happy that there were bands corrresponding to the sizes of my inserts. Their identities were confirmed from DNA sequencing.

These are the possible mistakes for failure in ligation experiment.
1. Ligase is usually dissolved in glycerol. Presence of >10% glycerol in the reaction mixture may inhibit ligase. In 10 ul of reaction mixture, 0.9ul was added instead of 1ul and the pipette tip was drained off excess ligase that might be adhering outside the tip.

2. Make sure the ligation buffer is new or used one is always kept on ice and stored at -20 degree celsius immediately after use since ligase requires ATP for ligating.

3. Vector and insert must be rid of ethanol since ethanol inhibits ligase too.

4. Calculate the molar ratio according to product information sheet. My mistake was that I followed others' recipes blindly. Alternatively, look at the formula below by Cranenburgh (2004) for calculating the volumetric ratios required in a ligation reaction. The T volume (represents the combined volume of vector and insert solutions e.g. T is 8ul in a 10ul reaction with 1 ul each of ligase and 10x buffer) can be scaled up accordingly.











Reference
Cranenburg R.M. (2004) An equation for calculating the volumetric ratios required in a ligation reaction. Appl. Microbiol. Biotechnol. 65, 200-202.

Too many distractions at night

Now that I have cable tv, my nights have been spent watching shows from the time I got back to the apartment till 11pm latest, unless it was the weekend which would be 1am.

Further, I have five books on hand. I am almost done with The Measure of a Man: A Spiritual Autobiography where the famous black actor, Sidney Poitier shared about his life growing up in the Bahamas to surviving as a black actor under the prejudices against colored people at that time in Hollywood till the present. It was written addressing to the reader, so I could relate to what he was conveying.

I am also reading On Call in Hell: A Doctor's Iraq War Story by Richard Jadick with Thomas Hayden. No point for guessing it right about the content of the book from its title.

The other three are Everyman's Library Pocket Poets by Robert Frost (a gift from Tom) who epitomized an American poet, Canaan by Donald McCaig and lastly Intuition which touches on the world of labs and researchers (on loan from my landlord).

In between restless nights, I would solve two Soduku puzzles to make my mind tired so I can sleep.

These past two days, I suffered from a bad stiff neck. It started with the right side yesterday. But today, the pain had spread to both sides. I am not moving my neck much. You can imagine how funny I am when I had to turn my whole body instead of just my neck to look at something. Sigh, hope it will go away quickly. I am sticking the medical patch to relief the pain. Vladimir suggested that I used a hair dryer to blow on the affected part.

Happy and blessed Easter!

After 40 days of observance and reflection (Lent season) beginning from Ash Wednesday which I did not practised religiously, it culminated in today's celebration of Jesus' living again.

His resurrection symbolises light in the darkness, hope for the despair, life after death. Easter happened 2000 years ago but is still happening in our daily lives when we love one another as He commanded.

Cherry blossom at DC (Episode 2)

Last year around this time, I went to see the cherry blossom with Greg at Washington D.C. but most of the thousand-odd trees had shed their beautiful blossoms.

This year, I am determined to see the Tidal Basin in full bloom. The peak bloom forecast for 2007 was Apr 3 - 5 from the Official Website of The National Cherry Blossom Festival. After my experience last year with bad weather and mistiming, I consulted with the weather forecast and decided Apr 3 (Tue) was the best day to go since it was sunny and temperature hovering around 70 Fahrenheit.

It was a good decision since it rained today (Apr 4). Greg initially planned to go today but I did not want to be caught in the rain. Also, the rain would cause the blossoms to fall.

On Apr 2, which was Greg's birthday, he invited us over to his home to watch the final in national college basketball between University of Florida and Ohio State University. Thereafter, Greg and I decided to drive to DC at 12 midnight to avoid the 4am rush towards DC.

We reached DC around 2am, Apr 3 and spent a good deal of time searching for free or all-day-long parking as all the parking lots had 2hr limit. Eventually, we found one but it was closed and we just parked right outside a bank at the intersection of 11th and E streets. (I was thinking what would happen if there was a bank robbery and we would be suspected as accomplices.) Greg brought along pillows and throw overs. This was the first time I slept in a car in USA. I was tired but woke up on every little sound. My survival instinct kicked in to check there was no danger before dozing off again. I suggested to Greg that we should sleep under starlit night at camp site instead of street lamps along a road.

Around 6am, the garage was opened and we parked the car. Morning was cold and we were not dressed warmly. Anyway, we reached the Tidal Basin and this time round, our effort was not in vain. The whole lake was surrounded by blooming cherry blossoms. What a magnificent sight to behold. Some of the trees were already shedding their blossoms which looked like snow flakes. The petals formed an interesting abstract art piece on the water too.

With 1 gb CF card, I was shooting away till I ended up with 161 photos. Taking good pictures at dawn was challenging with the low light intensity.

Anyway, I went back to work in the afternoon, feeling tired but satisfied!

Specially selected cherry blossom photos are available here.