Week 1 Diary

Day 1

-11am arrived at the Lab and given my intro. tour etc. The lab is very different from the ones I am used to in my A Level Science lessons:

The size of the test tubes I will be using!

The size of the test tubes I will be using!

The fridge.

The fridge.

-met with my supervisor and we talked about the work to be done.
– had the safety talk – there are a clearly defined set of procedures to keep everyone safe. I noticed a bottle of 5M sodium hydroxide in the chemical room, and released why these are necessary. At school we never use more that 0.1M, because it is highly corrosive!
Protective Equipment...

First day over I have a bench and I know my way round the lab – I’m ready to begin working.

Day 2
-template production
In order to discover which genes are present in which cells, we select the genes that we wish to find and produce them in a form that we can use for this means (a probe). Pre selected Plasmids or small and circular pieces of DNA found in bacteria contain the gene we want to “probe” for and enable us to make RNA probes that we will insert into the embryo cells we will examine.

The first step in making a probe is producing a template from which it can be copied. A template is a linear piece of DNA (i.e. not circular like a plasmid) which contains a sequence of bases that only codes for one gene, with a few extra bases on either end (see diagram).

This template is required to act as an intermediate between the plasmid and the probe. We cannot simply copy the gene from the plasmid into probe form because the enzyme which copies the gene in the plasmid will include the rest of the sequence that is included in the plasmid; this is because they often code for more than one gene (see point 1 in diagram). For our purposes we only require the sequence of the gene we are testing for to be present in the probe.

Making a Template
We have to “cut” the gene out of the plasmid to isolate it. However, I say cut but in effect we use the process of replication to make many copies of the gene:

1 We separate the two DNA strands so that the base sequences can be copied. This means heating the strands to break the hydrogen bonds hold the two parts of the ladder together.

2 We insert primers at the start of the gene on one of the strands, and another at the end of the other strand, which tell the DNA polymerase enzyme where to start or stop reading the code whilst transcribing the sequence (see point 3 in diagram).

3 We incubate the mixture which allows the enzymes to work efficiently. After the first replication, the two original DNA strands will remain, containing the primers, let’s call them the long strands. Two new shorter strands will be also made, in which the sequence now begins or ends at the point the primer is located because this is where the enzyme began or finished transcribing the gene (see point 4 in diagram). We will call these the short strands. After the second replication, the original longs strands are copied producing two short strands as before. The two short strands are also copied, so producing two more short strands. This means that after the second replication, there are two long strands and four short strands. In this way, if replication continues, many more short strands are made than long strands, and it is the short strands that we want.

A diagram to explain the steps in PCR.

A diagram to explain the steps in PCR.

Why do we want the short strands?
When the short strands bind together, they produce the template. Some of the short strands start at the beginning of the gene, but the sequence continues after the end of the gene (see point 4 in diagram). Other short strands do not start at the beginning of the gene, but they do stop at the end of the gene. This is due to the position of the primers. If two of the short strands bind, they can only bind in the region of the gene, because this is the only area where the sequences will be complimentary (see point 5 indiagram). And this is how we make templates of our gene.

In practical terms, this involved mixing the plasmids, primers, DNA polymerase and a few other chemicals together in a test tube then placing them in a piece of equipment for a Polymerase Chain Reaction (PCR). We can set this to maintain different temperatures inside for different periods of time. This is necessary to heat up the test tubes to separate the strands then adjust the temperature for the DNA polymerase to work efficiently and then cool to a temperature at which the DNA strands will reform.

Machine for PCR.

Machine for PCR.

Finally, to check it has worked, we run each mixture containing the templates on an agarose gel. In a small gel tank (see picture), we insert each sample into a separate indent in the gel (shown as a white rectangle in my results image). This is where the DNA originated. We then applied a current across the gel, which was in a buffer solution to act as an electrolyte and conduct electricity so that opposite ends of the gel became oppositely charged. DNA becomes negatively charged in solutions because it is an acid and so by definition releases H+ ions in water. Therefore, it moves towards the positive end of the gel. The distance it moves in a given time depends on its mass, so that the heavier the DNA molecule, the shorter the distance it will travel. This mass in turn depends on the length of the sequence because if the strand is longer, there will be more bases contributing to more mass. If the entire DNA is the same length, because the templates are of the same genes and of the same number of base pairs, it will all travel the same distance under these conditions and will form a visible band some distance away from where it originated.

Gel Tank.

Gel Tank.

These bands become visible under UV light because we add a chemical to each sample that fluoresces in UV light.

As can be seen in my results (which I will add on Monday), the second and fourth columns show that these templates were successful. The third column on the other hand was less so because there are several bands indicating several lengths of DNA, and not one highly concentrated area of template of the same length and same sequence.

Finally, we purified the templates, to make the DNA in the mixtures more concentrated. This involved filtering everything but the DNA and centrifuging the mixture to remove the rest of the liquid, and then re-dissolving the DNA in a smaller volume of liquid, in a process called PCR purification.

Day 3
-converting the templates to probes
-preparing the embryos
-washing the embryos

Converting the templates to probes
The templates are the pieces of DNA containing the gene that we isolated and amplified yesterday. Today we needed them in RNA form for them to bind with the messenger RNA inside the cells. We also need this RNA to contain the chemical which will be used in the signalling and staining stage to mark the cells containing the gene. So, to do this we need to convert the templates into RNA probes, so called because they probe for the gene we are looking for.

Several chemicals had to be added together for the conversion to occur. These included an RNA labelling mix, which contained the nucleotides (which hold the bases and chemical labels), the template and an RNA polymerase enzyme, which was specific to the templates and copied the template to make new RNA strands using the nucleotides we provided. We also should have added an RNase inhibitor, which basically inhibits the activity of any enzymes which serve to break down RNA and would try to break down the probes as they were created. These enzymes could contaminate the mixture by transfer from our skin, because we secrete these enzymes to destroy virus RNA which often use this as the molecule containing their genetic code.

Next, we incubated the mixture at 37 degrees Celsius for the enzymes to work efficiently and then we ran some of the sample on an agarose gel again to check the new probes had been made effectively.

Hot water bath for incubation.

Hot water bath for incubation.

These are the results (which I will add on Monday):

The bolder ones have worked the best and the weaker ones are still useable, but we will need to add greater volumes of the probe to the embryo to make up for the reduced concentration.

Then, to remove any templates which may interfere with our results once we add the probe to the embryos we added some DNase1, an enzyme, which is RNase free, to break down the templates made of DNA but without affecting the RNA probes. Again the mixtures had to be incubated for the enzymes. Finally, we used a form of chromatography to purify the probes, which involved filtering the mixture through some special paper which only allows the larger molecules through. Therefore, the larger RNA molecules can pass through and the smaller nucleotides collect on the paper, when centrifuged. At last, the probes are ready!

The other task of the day: start preparing the embryos. I say start because it is a very long progress (see image of protocol). We first examined the embryos under the microscope to select those at stages 9 and 12 and to remove some of the membrane attached to them. Next comes the washing. First we wash them in PBS (phosphate buffer saline). Then we wash them in methanol which makes the lipids (fats), particularly in the cell membranes much more permeable, because cells do not easily take up RNA and we need the RNA probes to be able to penetrate into the cells to reach the messenger RNA. Then we wash them in hydrogen peroxide dissolved in methanol to bleach them white. Then methanol again and we were finished for the day, but not with the washing!

The pipetting tips which I use when washing the embryos: sterile equipment is important to avoid contamination.

The pipetting tips which I use when washing the embryos: sterile equipment is important to avoid contamination.

Day 4
-embryo washing
The day started with more embryo washing, in methanol again. Then it was PBT, and finally to the exciting bit that all the preparation was for: the hybridisation. In this sense, hybridisation means the binding of complimentary base pairs in RNA, or DNA. In this case it was between the probe and the messenger RNA, in the region where the gene is expressed. It was time to mix the probe with the embryo so that this binding could occur. First, we prepared the embryos in a pre-hybridisation mix, and then added the probes within a hybridisation mix, using different concentrations depending on the results of the gel run, remember? Finally, we left the embryos in the hybridisation mix to incubate overnight, so the probe could get to work.

Day 5
Guess what? More washing, but with a different purpose this time. In the afternoon, we planned to add the antibody to the embryos. Their role is to bind to any of those chemicals that are attached to the probe. They have an enzyme attached to them that will convert a yellow substrate blue when we add it later in the process. The antibodies will bind to the chemical regardless of whether the chemical in the probe is bound to messenger RNA or not. Therefore, we needed to remove the fluid containing the probe that was surrounding the embryo and then rinse them many times over in various solutions including Solution X (sounds scary, I know) and MABT. This was to ensure all the probes that are not bound to messenger RNA are washed away, so that the antibody does not bind with free RNA to give a false positive result, or background.

Some of the washing solutions I have been using.

Some of the washing solutions I have been using.

Once we had washed the embryos enough times to be confident that most of the free probes had been removed, it was time to add the antibody. This antibody has an enzyme attached to it which will convert the yellow substrate to blue later-on in the experiment to stain the positive cells blue.

Finally, any free antibodies that were not attached to the probe also had to be washed away, again so that they would not give a positive result or excessive background. And with that, I was done for the first week already!

End of week one!
One week on and I know so much more about molecular biology, the protocol of scientific research, practical skills and the application of my A level knowledge. I have really enjoyed the challenge of working through problems and questions that the project has presented. It has also been interesting to progress from practical in-school science lessons to working with the tiny volumes in molecular biology and the accuracy and sterility needed in practical procedures that I didn’t know existed before. I am very grateful for this fantastic opportunity and next week will bring even more.

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