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.

Some lab updates

So its been a while since I had updates from the lab. The main reason is that things are going slow and I don't spend that much time there.

Nonetheless, few updates and some images. We finally got our very own ice-buckets (for some reason our lab-ware supplier had these in backorder for two months).

We are also testing a PCR machine. We got an offer to buy a machine from a reletatively unknown vendor that has good specs. To get a sense for it, we got a demo machine installed in our lab.

One of the nice features of this PCR machine is that has a detachable block, and so it can be either a 96-well block (for a full plate PCR or many reactions) or two 48-well blocks, allowing to run two reactions in parallel.

Next week we will get another machine for a demo, and then we will decide which one to keep.


As part of our work we often have to grow bacteria or yeast in colonies. When starting with very small amount of cells that can grow on the media, we often want to spread them on the plate. The common way of doing so is with a bunch of sterile beads. You pour some onto the agar

and then add the bateria dilluted in water (or liquid media)

and shake well.

After wards we discard the beads into a collection bottle for recycling, and move the plate to incubator for overnight growth.

Running our first PCR

Following our (stellar) success in running a mini-prep yesterday, Avital and I moved on to run a PCR on the plasmids we extracted.

If you do not know, the PCR (polymerase chain reaction) is a reaction in which we use DNA-polymerase, and enzyme that synthesizes DNA to be the reverse complement of a single strand DNA already in the mix. By moving between synthesis stages and "melting" stages where the DNA is separated into single strands, this reaction performs repeated synthesis of DNA. If we start with a single double strand, we get two after one round of amplification, four after two rounds, and so on, leading to an exponential increase in the number of copies.

The key to make a PCR reaction work is that the DNA polymerase has to have a small piece of double stranded DNA to start with (so it extends that piece). This allows us to control the reaction by introducing a sequence called a "primer" that hybridizes to a sequence it is complementary to. Once this happens, the DNA polymerase can perform its reaction. By using a pair of forward and reverse primers we can get synthesis on both strands.


The main work for us was taking the primers that we ordered and making them ready for use. This meant diluting them in very clean water (distilled, steralized, and then filtered) to a known concentration, and then preparing "work" tubes with lower concentration.

After, organizing all the primers (which took most of the morning), we moved to planing the actual reaction. This meant calculating metling temperature and designing the PCR mix.

(Avital calculating volumes in PCR mix)

Once all of the planning was done, the actual work was pretty quick. We created PCR mix, loaded the proper reaction components onto small PCR test tubes, and went to the neighbor's lab to run the reaction.

 

  

In setting up a PCR reaction, we essentially program a machince that can heat/cool the PCR tubes in a predefined schedule. The trick is that at high temperature (98C), the double stranded DNA "melt" into single strands. At lower temperature, the primers and the DNA template will anneal together. Finally, in an intermediate temperature of 72C  the special polymerase we are using (TAQ polymerase) is active and performs synthesis.