Posted by: ericmjl | November 7, 2009

Following some cleanup…

So now that we’re back from the Jamboree with a GOLD medal, we set about cleaning up Roza’s bench so that we could give it back to her. As Amelia cleaned up the plates, this particular one surfaced…

 

IMG_0301

Late-night salvo

Apparently, I had plated cells on the wrong antibiotic plate (supposed to be Chlor, plated on Amp-Tet). These cells had been recovering for 6 hours (don’t ask why).

Once I realized my mistake, I took the pipette, sucked back whatever I could of the 500µL, plated down that volume onto the correct antibiotic plate, and hoped that it’d work. Apparently it did. And so we have this successful plate.

Two wrongs, perhaps, do make a right.

 

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Posted by: Calvin C | October 9, 2009

First iGEM Club 2009/10 Icebreaker

Long time no see!

Sorry about disappearing for the last two months.  We all have been very busy finishing our experiments and analyzing our data and results.  We had some poor results about a month ago…but everything turned out ok after we found the culprit behind our failures.

We had our first iGEM club 2009/10 event today.  It was an iGEM Introduction presentation followed by an icebreaker with the new club members.  We had a good turned out.  There were approximately 30 new comers, but that was only because people were busy studying. (please note that there was an organic chemistry midterm that evening and a microbiology exam the next day).  We will be expecting an even larger turn out at our first journal club meeting.

Amelia made a very emotional speech today.  She spoke from the bottom of her heart about how she loves iGEM and all the people she worked (and pretty much lived) with this summer.  Go Amelia!

So we are all preparing for the Jamboree now.  It is in less than a month.  Although we got some good results, we still have a lot of work to do!

Posted by: ericmjl | August 15, 2009

3M Sodium Acetate

As part of our lab maintenance duties, I spent today testing the new miniprep reagents made yesterday. Since the yield (141.8 ng/µL) and purity (1.69) were not up to scratch, I decided to make a fresh batch of reagents.

Halfway through, I realized I needed 3M sodium acetate, pH 5.5 So at first, I thought, why not make the 3M solution, top up to 40 mL, then pH using HCl? Not a bad idea at first glance, and I tried it – and my yield and purity didn’t improve much (241.7 ng/µL; 1.77).

So I sat down and thought about this problem. It wasn’t right to introduce the counter ion Cl- into the solution, and that may have been the cause of much chagrin. Thus, I decided to try making the solution the other way – using acetic acid and then raising the pH to 5.5 using NaOH. However, no matter how I tried to calculate it, I couldn’t recall my Analytical Chem (CHEM211) concepts for calculating a titration. The volumes I had attempted to calculate were ridiculously large too – 17 mL of 1M NaOH?! That sounded like too much. How about using 6M rather than glacial acetic acid instead? That cut the volume, but would be too troublesome.

3 scrap sheets of paper and 2 hours later, I gave up and decided to look online. And bingo, the revelation came – 12.31g sodium acetate + water to 35 or 40 mL, then lower pH using glacial acetic acid, and then top up to 50 mL. I had been using the right reagents the wrong way. However, after 3 hours of exposure to glacial acetic acid, with an irritated nose, throat and personality, the yield went up to 392.03 ng/µL with a purity of 1.85. Purer, but not much more encouraging since I usually get yields of 2000 ng/µL, rather than 400 ng/µL, from 1.5 mL of culture.

Perhaps I am a biologist, not a chemist.

Update: 5 minutes after writing this post, I decided to just type up the protocol anyways. And that’s where I caught where I may have gone wrong – I didn’t adjust the pH of the Tris-Cl buffer to 8.0. Grr, I really need a protocol to start with.

Posted by: ericmjl | August 6, 2009

Molecular implementation of simple logic programs

http://www.nature.com/nnano/journal/vaop/ncurrent/full/nnano.2009.203.html

One day, I’m gonna present this at Journal Club. 😉

Posted by: Calvin C | July 24, 2009

We are more than lab geeks

We have all seen how great some of us are in the lab.  A few team members can recall most of the BioBrick part numbers off the top of their heads, a couple can answer most of my lab questions faster than Google, and one can even pipette nearly twice as fast as I can.  However, their awesome skills do not exist only in the lab, but many of them have some interesting talents and hobbies that might be surprising to you.

Lets start by talking about some of the sports that we do.  The most obvious one is the hardcore biker we have.  While most of us Vancouverites love to hop on our cars to go around town, we have a team member who takes his bike wherever he goes.  Whether it is a short ride around campus or a trip to downtown, he is always doing his part to decrease CO2 emission by riding his bike.

Although not everyone on the team can handle biking everywhere, most of the team members are fairly physically fit (this is essential for late night work in the lab).  A few of us go to the gym on a regular basis, one of us is a life guard and a number of us are rock climbers.  Sometimes we have a hard time stopping ourselves from turning the Michael Smith Lab (MSL) into a jungle gym.

For anyone who has been to the MSL, you probably have noticed that the UBC campus security office is directly below us.  Having security so close to the lab may seem reasonable since there are some expensive equipment and valuable research data and personnel in the building.  Although it may not seem obvious, a few of us are highly capable of protecting ourselves and our valuable bacteria.  For hand-to-hand combat, a few of us are trained in Hapkido and Chinese martial arts.  When the fights involve weapons, we all turn to our Cloning Queen who has a black belt in Kendo and is highly skilled in archery.  Unfortunately for her (and lucky for the rest of us), MSL does not allow lab personnel to carry a katana to work.

In addition to sports, most of our team members are highly involved in volunteering.  A few of us have helped out in the hospitals, research centres, campus clubs and departmental undergrad societies.  One of us has taken the brave assignment of chasing, capturing and caring for wild animals in need.  Two of us have started volunteering at the crisis line recently; they will be very important later on!  Another two members have been on expeditions out of the country earlier this summer; one went up to the Arctic to explore the wild life and the other went down to Ecuador to build schools for the children.  How admirable!

In conclusion, I hope that this blog let you know about our team a bit more.  I am sure after another month with my teammates, I will be able to write a lot more about them.

Posted by: ericmjl | July 14, 2009

My work went down the drain.

Literally, it went down the drain. But it’s okay, that was just one digest out of 9, which is easily repeated.

The harder part is repeating the cPCR that yielded some good results, and some missing results (no colonies, grrr).

Recently, a friend reflected to me that because we were working late into the night, one day one of us is going to get the Nobel prize.

Well, I wonder. It’s not necessarily the scientists who toil into the night who win the Nobel prize. Nobel prizes are given to the most “inspired” (as opposed to “inspiring”) scientists, scientists who make a breakthrough. Need an example? Think Kary Mullis. 😉

Posted by: ericmjl | July 11, 2009

A Promising Outlook

At least, it’s a promising outlook for myself. I ran a PCR today, and even though there were contaminant bands in the water (which I expected, given that we haven’t changed our primer set yet), and the PCR on the parts I had identified, transformed and miniprepped (with Mark, Karen and Amelia’s help) indicated that the band sizes were correct relative to one another, although I must say that the band sizes weren’t correct relative to the 1 kb or 100 bp ladder that we had. Perhaps it’s the amount of DNA that I loaded. Amelia and Heather seem to have run into this problem before, and sequencing results indicated that they were fine, so I guess I should be fine too.

In any case, it sounds much more promising than the previous experiments that I had conducted, because I kept getting negative results, and at least this set of results are pseudo-positive.

Posted by: ericmjl | July 8, 2009

ssDNA – 3 weeks facing the scourge

So for the past 3 weeks, I have been trying to clone a 100-ish b.p. fragment of DNA into our ampicillin construction plasmids. For 3 weeks, I have faced endless frustration in the face of failure. Today’s revelation about the ultramers that we ordered from IDT was simply a relief off my back.

It turns out that the ultramers are ssDNA strands, not dsDNA strands, so naturally they weren’t cutting well. (I’m half crying and half laughing at my own stupidity as I write this.) That’s why for the longest time I was getting few transformants (1 or 2 colonies a plate), and even those transformants carried a plasmid with little sequence homology to the original inserts. No wonder things were harrowingly frustrating. No wonder Amelia also failed to clone the lock/key/control lock in (I was expecting her to succeed where I had failed).

Now that I know, I guess I needn’t be depressed about my inability to clone that 100-ish b.p. sequence into an Amp construction plasmid. I guess I should move on – after all, my digests certainly look good, and my ability to ligate seems fine.

Onward I go, moving onto the next step – trying to do an actual assembly of new stuff, really exciting stuff.

Update: To rectify this problem, we’ll PCR up the ssDNA using the fresh new primers that we’ve got. That should solve the problem. A sequence check should help us make sure that the parts are fine.

Posted by: ericmjl | June 23, 2009

Something’s Fishy

For the past week, I have been attempting to clone an approx. 100 bp insert into our ampicillin construction plasmids (approx. 2.1 kb), and this has proved excruciatingly frustrating. After initially assuming that the transformation went fine and good (because I had obtained colonies), I digested the mini-prepped plasmids from those colonies and screened them via a restriction digest on the plasmids before running a gel of the said digests, only to find that the band sizes were not 100 bp. and 2.1 kb. Rather, I encountered a myriad of band sizes, from 300 bp to 450 bp to 1.8 kb to 2.2 kb, but none of the 100 bp and 2.1 kb sizes that I had expected.

In order to troubleshoot this, I took a backup set of ligations (which were an overnight extension of the 1 hr ligation which I had originally carried out), and did the whole motion of transforming, mini-prepping, and digesting, only to find even more bad bands. That made for a harrowing weekend of frustration in the lab. Concatamers are fairly impossible, as the EcoRI and PstI cuts don’t have compatible overhangs. A gel of a fresh set of digests + ligations yielded blank bands, which means that I just have to transform and have faith that everything goes well.

Meanwhile, the original minipreps have been sent for sequencing. I just hope that after doing a sequence alignment that everything looks okay. Then I can stop everything else and running into this brick wall.

Anyways, back to the transformation.

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