Short renovations update

The call for proposals for A/C and installation systems has gone out this week. Today we saw the building supervisors lead potential contractors in a site tour. Hopefully with in two weeks we will have the proposals in and decision on the chosen contractor.

Singer RoToR troubles (follow up)

Yesterday I wrote about the Singer going into problematic state. I wrote a long email to support at Singer Instruments, and connected over the internet with Ian (who installed the machine) to try to fix the problem.

This was quite interesting as he connected by remote control and enabled windows that reported on the inner working of the software (sensor values and such). It took us a while, but he established that the sensor on the gripper head are not working properly and thus it cannot lift up pads.


To make a long story short, we found out that when we tried to clean the gripper head we had to disconnect the cables leading to it, and in doing so we tried to untwist them and managed to tear up the wires.

This raised a problem of how to fix the cable. Ian suggested he will ship a new one, and I promised to look for help from local electricians. I called Danny at the System Group here in HUJI, and he and Dima showed up with solder tool. In no time Dima managed to reconnect the plug and it looked like new.

We called Ian again and went through the test procedures. This time things were working fine. He tuned the parameters to make the robot less prone to the intermident problems it had last week.

In conclusion the Singer is back online, and we hope it will continue to behave nicely.

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.

 

Mini-preps and Gels

As ours is a new lab, each one of our baby steps in molecular biology is a small accomplishment. Today, we run our first mini-prep. Yey!

The mini-prep procedure extracts plasmid DNA (small rings of double stranded DNA) from bacteria. We use the bacteria for making many copies of the plasmid of interest. Basically, we feed it and in return get amplification of small amount of plasmids into large quantities we can work with.

While running a mini-prep is day-to-day routine in many labs, doing it the first required to make sure we have all the ingredients. It was also a first time for me in teaching a protocol. So Avital had to see me learn how to teach...

Ayelet and Avital grew the bacteria overnight from stock we had in our refrigerator. This morning Avital and I started by centrifuging the bacteria into small pellets, and then remove the media by pipetting.

 

(photos courtesy of Ruty)

We then started with the kit instructions for the protocol. As you can see, the little card summarizes the steps.

 

The heart of the kits are these mini filter tubes that can bind DNA under certain conditions and release it when washed with a specific buffer. This means that during the first washing stages we run the supernatant from the cells and the DNA binds to the filter while all other molecules pass through, and then in the last wash the DNA is released into the small sample tube.

  

To carry these steps we use a small centrifuge. The centrifugal force serves to drive all liquid from the top chamber to the tube below, moving it through the filter. 

 

Once we finished the mini-prep we wanted to ensure that it worked. First, off as a test of the plasmid carrying bacteria that we were using, and second to teach ourselves the careful steps in checking our procedure. 

We did two test. The first is to run the samples through a spectrophotometer. Unlike the use of spectrophotometer for measuring cell density, here we use it to check that we get absorption at the right wavelength (260nm) which indicate that we have DNA. Since the final sample is very small (50 microliters), we used a cool device called NanoDrop that is a spectro-photometer on a drop of 1 microliter. See the liquid at the tip of the pipette here:

The NanoDrop showed that all of our samples had very nice spectra, suggesting no contamination. It also computed for us the concentration of DNA in the samples.

Next, we wanted to make sure we didn't get any genomic DNA or other stuff. For this, we run a gel electrophorsis. We opened the gel device from its wraps, prepared TAE buffer, and "cooked" agarose gel in our lab microwave. We then poured into the mold, using a "comb" to create small wells in the gel.

After the gel solidified, we filled the device with buffer. To load the gel, we needed to combine 1 microliter of sample with a dye that will highlight the DNA. Ayelet taught us a neat trick to prepare the mix using small droplets on a parafilm.

 

We then used a pipette to load each of these small drops into one of the "wells" created by the comb in the gel mold. This meant aiming the tip into a small rectangle in a translucent gel within a water bath.

Once  the gel is loaded, we connected the bath to electrodes, and run 120 volts through it. The bubbles from the electrodes show that proper electric field is established.

To our delight, the gels result showed nice clear bands in our samples.

Singer RoToR troubles

I promised to write of our successes and setbacks. Well, we had a bit of a setback the last week.

Remember the Singer RoToR colony handling robot that we are so proud of? Well, we (that is Ayelet) started using it for real last week and we started having problems. It started with small issues, where one of the plate lifting arm was not functioning well. Whenever this happened, the run had to be aborted. Apparently, the abort-run software function did not do a complete reset, as after the reset the robot worked as though it had a pad on, and crashed its head into a plate with 384 colonies. Needless to say that the plate is not usable, but more seriously the whole pad-loading head was full of yeast and agar.

Today we decided to try to go through the steps slowly and see if the problem repeats itself. We found that the robot was not succeeding in going through the first initialization where it counts the number of pads in the pad drawer.

We contacted the company and hopefully they will get back to us soon.

Work order issued

As one small step toward our new permanent lab, today the university issued a "work order" for the constructor who is renovating the new room.

I am not sure when they will start actual work, but hopefully soon we will have pictures of handymen in action.

Back from vacation

The last week I went on vacation with the extended family to Samoens, France part of the Grand Massif ski area. We enjoyed the slopes,

the wine

and also the fresh snow...

In the meantime the lab was busy. Ayelet, Avital, Ruty and Ohad worked and managed experiments with the Microscope and the FACS (more on these during the week).

We also had some new arrivals, the most impressive one is the MasterClave - a device for preparing and pouring agar media.