Week 3 Diary

Day 10
Returning to the lab after two weeks off could have been strange, but it all came back as soon as I opened the door to the familiar smells of the lab. With most of the photographs taken before the holiday, we would now be able to move on to the next stage of the project, where we would be investigating new genes, specifically Neurogenins 1, 2 and 3.

The first part of this stage is, as before, to make templates and then probes which can bind to messenger RNA in chick embryos during in situ hybridisations. Unfortunately, in the two weeks practical time I have left in the lab, a second set of in situs may not be possible. I may have to hand the probes that I make over to some of the others who work in the lab to do the in situs, and they will keep me updated with the results.

So, today I made the templates, as before, performing PCR with water, PWO (which contains the polymerase enzyme and the nucleotides), genomic chicken DNA and primers. We had to order the primers from a supplier to bind to the regions on either side of the non-coding part of the paralog genes we are using.

What are paralogs? In the early history of life, the genomes of ancient ancestors have replicated themselves, so that after replication, the genomes contained two copies of each gene. Several of these replications have resulted in the genome of many descendent species having several copies of each gene. Independent mutations of these genes have made them slightly different from each other and we know them as paralogs. Paralogs are similar to each other in terms of base sequence and role.

Why the non-coding region? The Neurogenin genes are paralogs of each other, so they have very similar base sequences, particularly in the coding region, the introns. The non-coding regions, the exons, are more specific to each paralog, so probes that bind to these regions will give more reliable results. Otherwise, the probes may be very similar and bind to messenger RNA containing the other paralogs.

Before the holiday we determined and then ordered the primers we needed. This involved agreeing the best region of the gene for the primer to bind with. We sent the required DNA sequences to a biological supplier that produces primers. The primers soon arrived in the post, although the tubes containing the material we needed initially appear empty. According to the rules of Molecular Biology, “An empty tube is never empty”, there was no need to panic. We had to trust that the DNA primers really were in there and added water to produce solutions.

The forward and reverse primers with the PWO, water and genomic chicken DNA went into one of the smallest test tubes I expect to come across, and then straight into the PCR machine. The PCR is used to set different temperature conditions for different time periods. These different temperatures are for the DNA strands to break, the DNA polymerase to copy the genomic DNA within the region set by the primers and then for the DNA strands to reform giving the template. The cycle of temperatures repeats many times so that the template is produced exponentially.

We did this PCR process twice today, first using primers to produce a long template and then again with some primers that bind within this long template to produce another, shorter template from this. This basically means the exponential production of the template occurs twice, so we produce a large volume of the template.

The PCRs take 3 hours each, so, there was plenty of time for more photographs of the chick embryos. The second PCR has been left to go overnight, ready to be run on gel tomorrow morning.

Day 11

Today was a day of gel-running and centrifugation. The newly-made templates came out of the PCR machine and we ran a sample on an agarose gel (a bit like agar). This process of running on a gel separates DNA strands of different lengths. The shorter the DNA strand, the lighter its mass and so the further the distance it travels in the gel. This means that bands of DNA form where DNA of the same length collects. These bands are difficult to see with the naked eye but can be seen under a strong UV light due to the fluorescent ethidium bromide dye that we add to the gel.

Ethidium Bromide. Source: learning.covcollege.ac.uk

Ethidium Bromide. Source: learning.covcollege.ac.uk

We ran each of the templates in the gel, next to a marker sample of known lengths of DNA. The template ran the same distance as the marker sample of 400 nucleotides, confirming the length of the templates as 400 nucleotides, as we expected. This proved that the templates are the right size and we can assume that the correct section of the chicken genome has been amplified during the PCR.

However, there was one problem. Some of the forward and reverse primers that we put in the PCR mixture produced a second and unwanted band of DNA. These primers and any other DNA that was not the template, needed to be removed.

Removing this unwanted DNA meant running another gel, so that the short primers and the longer templates separated. After this, we shone a weak UV light onto the gel; weak because we didn’t want the UV to break any of the template DNA. Under the UV light, the DNA bands fluoresce in a bright orange colour. All that needed to be done then was to cut out the orange bands of the gel containing the template.

Next up, purifying these DNA templates. The gel sections that were cut out contained the templates in low concentrations, so we used a purifying and centrifugation process to increase the template concentration and remove the gel.

First, we melted the agarose gel by incubation in a hot water bath. This produced liquid agarose solution, containing dissolved template. Second, we transferred this solution into a centrifuging column, and added a buffer solution which causes DNA to stick to the white filter (see photograph). Centrifuging this mixture meant that the liquid (buffer solution and agarose) passed through into the collecting tube (see photograph). We can discard this liquid that collects in the collecting tube, because the DNA is safely stuck to the white filter in the column, which we keep. Then we repeat the process, but using a wash buffer instead, to wash through any left-over gel. Finally, we repeated it one more time with an elusion buffer, which un-sticks the DNA templates from the white filter, so that a concentrated sample of DNA filters into the collecting tube.

And just to check that this had all worked (you end up with a clear liquid that you hope contains enough DNA), we used UV spectroscopy. The UV spectroscopy involved shining a UV light through a sample of the template, and then measuring the wavelengths of the light that emerged after travelling through the sample. We expect DNA to absorb wavelengths of about 260 nm, and so when the emerging light was missing this wavelength, we new a high concentration of DNA was in the sample. The templates are a success!

Day 12

How to ensure the solution of templates is completely pure? As yet, we cannot be sure that it is. We know that the solution contains short fragments of DNA, many of which are the template, but we cannot guarantee that all are the template. The DNA polymerase may have also copied different sections of the genomic DNA at different sites to the neurogenin genes, thus giving small concentrations of these DNA fragments in the solution.

Since our neurogenin templates will be used to make probes which will again bind with messenger RNA in chick embryo cells, if there are other templates in the solution they may create probes of sequences other than that of the neurogenin genes that we are looking for. In that case, these alternative probes may bind with messenger RNA, because they are complementary to it, whilst the neurogenin gene is not actually activated in that cell thus giving a falsely positive result.

So, we have to use molecular cloning to make these templates pure.This is a process where a gene is inserted into a plasmid and then bacteria are encouraged to take up this plasmid and replicate it or clone it. Then, through a clever use of antibiotics and antibiotic resistance that I will explain later, it is possible to kill all the bacteria that have not taken up the plasmid and leaving colonies of bacteria, all of which contain copies of the plasmid. Samples are taken from the colonies and are grown in separate liquid culture solutions. Finally, plasmids within E. Coli bacteria, taken from each culture solution, can be extracted and sent off to be sequenced, so that we can know which solutions contain pure samples of the plasmid containing the desired gene.

Today, we started this process. We took plasmids containing two genes; one gene, for this purpose gene z, and another gene coding for resistance to the antibiotic Kanamycin. Then we added this together with the template and the enzyme BP clonase and incubated it. The enzyme recombined the template in place of gene z, in other words it “cut-out” gene z from the plasmid and put the template in its place. This produced a plasmid containing the template and the Kanamycin resistant gene.

Next we mixed the plasmids with the E. coli bacteria. Bacteria do not like to actively take up DNA, but they can do it so we first created favourable conditions to encourage the uptake of the plasmids. We incubated the bacteria in a culture solution with the plasmids and left them enough time to activate the genes within the plasmid and so to switch on their resistance to Kanamycin.

Now the bacteria were ready for transfer onto agar jelly, which contained the antibiotic. All the bacteria which had not taken up the plasmid were not resistant and so they died. This left a few individual bacteria, spaced out across the agar. We incubated these agar plates, so that these individual bacteria could replicate. E. coli divide every 20 minutes in the correct conditions, and so after we have left it for 20 hours, these colonies could have up to 2 to the power of 60 bacteria within them! We will have to see tomorrow whether the bacteria have replicated and if we have colonies.

Day 13

Results day, in both senses. I got my AS results and the photographs are finished so the results are in! All the chick embryos and their staining have been photographed ready for analysis on when and where the genes are activated in these chick  embryos. A first look has shown that some of the genes are expressed in the brain, while others only in the spinal chord section of the neural tube. Other genes are expressed much later, so may not be required to initiate neurogenesis, but rather maybe an important step within the differentiation process. Some of the genes may only be activated in neural crest cells, i.e. those that start as cells in the neural tube, as though they will become part of the spinal chord or brain but migrate outwards to become tissue such as the skull. There are some interesting possibilities posed by these photographs and further analysis for my project write-up will reveal more.

And, of course the other result of the day was whether there would be successful colonies. The good news is that we do have colonies, but the bad news is we only have them on the agar plates with neurogenin 1 and 2. The bacteria with the neurogenin 3 plasmid have replicated, but in a strange line that is not like the discrete colonies that we wanted. The reason we wanted discrete colonies is because we know that each colony is made up of identical bacteria, or clones, with exactly the same DNA sequences. If these colonies have merged, we do not know where one set of identical bacteria ends and the other begins when we take samples from them.

E. Coli colonies. Source: wikipedia.com

E. Coli colonies. Source: wikipedia.com

When the few surviving bacteria replicated, a colony gradually formed around them as they replicated. Every descendent of this bacteria has a copy of the plasmid with the template and the resistant gene. However, as I explained yesterday, not all of the templates are neurogenin templates, so some of the colonies will have a plasmid with an unwanted template.

However, this doesn’t matter because we have discrete colonies with different plasmids, so we have separated the plasmids into separate and pure samples. The next job therefore, was to isolate some of the colonies, in the hope that some will contain the plasmid that we want and will give us a pure plasmid sample.  We took samples from separate colonies and added them to separate liquid culture solutions to divide. This way, the bacteria will continue to replicate themselves and the plasmid, and we can guarantee that each culture solution contains bacteria with a plasmid containing only one type of template. Once we have extracted the plasmids from each solution, we can send them off for sequencing to know which solutions are a pure sample of a neurogenin template, and which are pure samples of alternative templates which we do not want. Clever, isn’t it? We have left the bacteria in these culture solutions to continue to replicate and tomorrow we will extract the plasmids.

Day 14

It was time to extract the plasmids from the bacteria. However, there had been a mistake. We had expected the mini-preps to contain cloudy solutions, full of E. coli bacteria but instead we found clear ones.  We therefore knew that the bacteria had not replicated as we expected. In fact, they may have all died. The most likely cause is that the culture solution contained the other antibiotic that is often used for this process: ampicillin. Of course, our bacteria only had resistance to kanamycin and so this antibiotic killed them off. On the bright side, we still have plenty of colonies left on the agar plates, so we will repeat the previous step and take samples from the colonies again.

With this mistake made, the rest of the day was for preparing the next set of chick embryos. We fixed them in 4% paraformaldehyde, ready for the next in situ hybridisations. These will be done by other researchers working in the lab, using the neurogenin probes we are making. As the results of these become available, I will try to post them on the blog.

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