Lab news - fixes and PCRs

Today we have quitely marked an important milestone. Itsik and Ami from the "fine mechanics shop" came to fix the Microscope, HyperCyt and KiNEDx rail to the tables. This means that from now we can assume that their relative positions with respect to each other are fixed (and thus the KiNEDx arm can return to the same position reliably). This is also an indication that we are happy with the temporary outline of devices we put down.

We were warned about the difficulties in drilling in the Terspa benchtop we have. It turned out that with a simple drill it was relatively easy to have holes in the benchtop. 

  

Ami threaded the holes, and then screwed in the equipment.

The HyperCyt is held in place by three machined blocks (two in front and one in the back). The Microscope had its holders that allow for screwing into the table and into the microscope body.

Finally the KiNEDx rail has holes in the rail itself, and it was screwed directly into the benchtop without external holders.

While Itsik and Ami worked in the Robotic room, we received a package.

Our new PCR thermocyclers. We got a deal on a pair of devices from Bio-Rad that combine 96-well head with two 48-well heads under a single controller. This was one of the missing large equipment on our "to get" list.

The army marches on its stomach...

... and science progresses on caffeine. Our newest addition to the lab is the new esspresso machine.

This was gift from Neotec to accompany the integration of the robotic facility.

This definitely improved our quality  of life and allowed us to discover new talents of our members. Without much practice, Ayelet created this nicely layered construction.

Blasting yeasts

In one of our projects we want to extract RNA from yeast for measurements using the nifty NanoString nCounter technology. One of the nice things about this assay is that it uses very small amounts of actual material to measure RNA quantities. Moreover, unlike other assays, we do not need to purify RNA before the assay, which saves a lot of headaches.

The challenge we posed to Assaf, who is about to do the experiment, is how to extract cellular material from a very small yeast sample. We actually need as few as 15,000 cells (just for reference, 1 microliter of happy growing yeast has about 1-2 x 10^7 cells). The problem is that yeast, like many other microorganisms, has a strong cell wall made from proteins. 

The classical way to break it is using mechanical action -- beating the yeast using small glass beads. This technique, however, does not scale down for small samples. And so, we decided to go with Plan B. Here we use an enzyme, Zymolase, that digests the cell wall. This creates spheroplasts -- cells without cell walls that are encapsulated by a thin membrane. We can then pop these open by diluting the sample with water with small amount of detergent.  This solution also includes protein denaturing agents that block any RNAses from breaking down the fragile RNA, and thus preserves our sample.

Assaf and Ayelet tried this technique, and it seems successful. The zymolase readily digested the cell wall and left with nice spheroplasts. Then when he dilluted these quickly disappeared. Assaf made a nice movie of this process under the microscope. What you see is a small drop with yeast spheroplasts, and midway through the movie this drop is dilluted.

Robotic Arm Integration (and other news)

The last week I was away on travel. During the week Shai (from Neotec) together with Avital & Assaf worked on robotic programing and hopefully we will have updates from that front.

In addtion, Udi and Yoram from Neotec manufactured a nice station for handoff between the Tecan Liquid Handling Robot and the KiNEDx robotic arm. Today Shai together with Amir and Moshe (also from neotec) worked on fine tuning the integration of the robotic arm into the Tecan control software.

At the end of the day we managed to film a demonstration in which using EvoWare (the Tecan control software) we can take a plate from the deck of the Tecan to the microscope/hypercyt and back. This means that we made a significant step toward integrating the system.

It is impressive to see the KiNEDx arm in action. Its movements are fast and fluid.

From the movie you will also notice that we hang the plastic curtains that surround the Tecan and keep it clean and sterile. We still need to tailor a hole for the KiNEDx to move through, so for now the enclosure is not complete.

Just to get a sense of the flurry of activity that led to these results, here is a time-lapse of part of that day.

HyperCyt and Python Scripts

Today we had a long day in the robotic room. Yesterday Arik from Neotec came to calibrate the find details of the liquid handling arms on the Tecan which solves some issues we encountered on Tuesday.

Today Shai, who is responsible for "smart" robotic application came. We had a general discussion about different ways of integrating software to the robot controlling software. Afterward he, Avital and Assaf sat down and implement some pythons scripts that call the robot and others that the robot can call.

It seemed that this was very fast pace study as they reported success, and managed to fancy pipetting procedures on the fly.

In the meantime, Ariel and Jenia tried to get the HyperCyt to work in the new location. We decided to move the peristaltic pump onto the hypercyt deck. This however resulted in the arm pushing the pump off when the device was initialized. This was annoying, especially since the pictures of the device showed the pump sitting exactly where we put it. 

We searched the manual high and low, and finally realized that one of the figures mentions an L-shaped piece that serves as a stopper in such a configuration.  We used this as evidence that this is the right solution and installed the stopper, which indeed solved the problem.

However, now the coordinates of the arm were totally off. And so we learned how to "teach" it where the different locations are. This involved moving the tip of the needle very slowly with mouse controls until it was in the right location.

In the end the system was working, and we even managed to film it. As you can see the sampling needle goes into each well in succession. This means that it creates alternating bubbles of media (+ cells) and air in the tube. If you look carefully in the movie you can see these tubes.

After lunch break and group meeting Jenia run his nifty analysis software to break the long FACs stream of events to specific wells. It worked like a charm and immediately gave him detailed summary of each well. The investment in this software was definitely worth while.

First steps with the robot

Today we had our first training session with the Tecan robot. We learned how to create a script for doing various steps (e.g., pipetting, moving plate from one device to another and such). The concepts were relatively simple and intuitive, and by lunch we had our first working protocol. It takes a plate, seed it from another plate. Move the plate to the shaker for a minute and then measures optical density.

After lunch we moved to more complex protocols that used multiple plates (e.g., when you want to run something on a large number of plates). At the end of the day we felt that we can start making use of this device.

We also had a our down moments, including a crash when the robot arm that moves plates decided to go to the first available position, which was not available....