A deep breath

Today Yuye is back from China. I don't know if he will be back at work or not but, he is back in the country. I am very happy about that.
I am really comfortable with all the QC duties that I have been doing.
BUT
Holy Cow! SO busy.
Working what amounts to a job and a half while going to school and being sick for months has not been a pleasant experience.
I have a lot of hope that once I feel less sick everything will be ok.

I am very proud of myself and I am looking forward to learning the rest of the PCR types now that Yuye is back. I am enjoying the feeling of being taken more seriously by co-workers now that I can solve a new type of problem. I am proud to be showing some of the things that I have learned to a co-worker who tried and failed to get into this co-op program.

I am very tired though.

An over due update

If you go over to my family blog http://blogofmissy.blogspot.com/2013/11/the-update.html you will see that I have been sick sick sick for the past 2 weeks.
Not the simple kind of sick that is over when its over but the more insidious kind of sick that involves tests and more tests and possibly lifelong medication.While it could certainly be worse its less than ideal for this class.
I have been so lucky in its timing because no major assignments have been due and I will be able to get in my co-op academic assessment on time.
BUT
I haven't been at work and I haven't been practicing my PCR. I feel like its all still rattling around in there so tomorrow I will assume my usual duties.
This return to work is coming none too soon as Yuye is leaving for China for a month on Thursday.

YIKES!
Wish me luck...

Here I am in my non-mom guise.

That is the door on one of the robots I am paid to kick around.

Great progress

This week, because Tony is back and the Hamilton technician is here, I have enough spare time to really focus on the QC training. It has made a huge difference.
This morning I was shown to a plate and told to come and ask if I had any questions. I made all the forms found and created the right reagents and templates, ran the PCR machine programs, analysed the results and did the gels by myself.
I definitely needed confirmation on the analysis step but got all but one well right.
Now I am learning the results phase with lots of minutia about which files to upload and where to put finished analysis. I feel great about this. The uploading is the final step and is the only one I cant do on my own (baring unforeseen disaster.).
I am not fast and I am not confident but I do think if I needed to I could now cover for someone in QC if they got sick tomorrow.
I think with another week of this amount of time, I wont dread the upcoming vacations.

Here is the RT2 and Methylation plate I got this morning.

A rough week

This week was supposed to be my big independence week. The hope was that I would be running PCR plates from start to finish on my own by the end of the week.
Unfortunately my friend and co-worker Tony has had a rather serious medical problem and is in the hospital.
That has changed by an order of magnitude the amount of free time I have to devote to cross-training.
I did actually run my own PCR plates twice this week and that feels a little like a miracle at this point.
I feel the upcoming vacations of all the many QC folks that are going to China in late October and early November looming over me like a tidal wave.
In other news my robots are all having personal meltdowns this week....
its not even a full moon.
My job right now is kicking robots and scurrying about trying to fill in big shoes.
I am stressed.
The good news is that Tony is OK and they will be letting him out of the hospital maybe as early as this weekend.
The other good new is that when I ran things they went well.
The bad news is I am just not ready to take over for anyone at this point and I am not sure that I will get enough time to practice before I am given that job.

Huzzah

I ran my first RT2 and Methylation plates on my own today. They worked well and I remembered all the steps. I also took my first whack at doing a gel myself. So far so good!
next week I will be in QC every day working on PCR and gles and hypothetically doing analysis too.
It is a good thing my other class is wrapping up. I am about to be completely out of spare time.

Yay!

chugging along

Last week was the end of quarter, which means in production, a very busy time. I hardly did anything with QC. Now its October and I am back in the saddle. Today I did 8 gel plates and got some big news.
I am being transferred to QC for between 2 weeks and 2 months.
This is going to be great for this class. I am going to spend a great deal of time following Yuye around in the next few weeks. Then in the beginning of November or the end of October I will be taking over several PCR duties.
I am excited and a little scared.
I am so glad to have the opportunity and proud that I am being trusted with it.
I am also anxious to not screw anything up.
Tomorrow will be my first day of heavy shadowing in preparation for my upcoming responsibilities.
Wish me luck.

Complementary Classes

I never imagined when I signed up for Forensic Biology how very useful it would be for this co-op class or for my work.
In the last few weeks we have been covering DNA analysis and PCR.
Not only am i getting a good hands on feel for the PCR process I am now, thanks to my class, getting a good hold on the whys and wherefores of my actions.
Here are a few conference posts from the last 2 weeks.
In each case I am expressing things learned only in the last few weeks.

PCR is the man made simulation and amplification of the natural process of mitosis.

DNA is doubled and doubled again until there are enough copies. The man made version of this mitosis is called Polymerase Chain Reaction.

Polymerase is an enzyme that causes DNA or RNA to form.

Chain refers to the cyclical and repeating nature of the operation

Reaction in this case illuminates the forced and controllable nature of the process.

As with DNA’s natural in cell cycle only small sections of the full DNA strand are replicated at any one time.

In normal Mitosis this copying is done through RNA replication.

I am going to crib from my own entry from a previous post here.

“The DNA in the center (the nucleus) pulls apart into two independent strands. Imagine a ladder in which the two long sides (strands in the case of DNA) pull apart. As the independent strands are separated by an enzyme called helicase, one strand, called the leading strand, is copied forwards and continuously and the other strand, called the lagging strand, is copied backward and in pieces. Proteins called DNA polymerases build new strands of DNA from the original (parental) strand serves as a template for the new strand. This will allows the new DNA structure to replicate exactly the original because each rung of the ladder (the bases or nucleotides called A,T,C or G aka. Adenine, Thymine, Cytosine and Guanine) attracts one very specific partner nucleotide and only that one.”

In the case of PCR, the action of helicase (that of breaking the strands apart) is preformed through simple heat. The heat is high enough to melt the DNA strands apart but not high enough to actually destroy the individual strands.

At this point as with Mitosis sections of the DNA strands are replicate. Unlike normal Mitosis or even normal RNA replication, very specific strands are desired. To eliminate the undesirable DNA, or even contamination, very specific primers are added to the mix.

Primers are prearranged nucleotides (ATGC’s) that are meant to match up only with the DNA sections the lab wants to see. These primers come in pairs. There is one primer for the beginning of the desired section and one for the ending.

As with normal DNA replication this process is very fast (about 30 sec.). In PCR repeated cycles are forced through repeated heating and cooling back down of the sample DNA.

My PCR runs usually take about 2 hours and can have up to 30 of these cycles.

The DNA replicates exponentially. I have attached a picture of what 6 cycles look like to give you an idea of how effective this process is. In a nutshell a single DNA can go from 1 double helix of DNA to over a billion in under 4 hours.

The second part of the question “What do the following mimic: Buffer, Taq polymerase. Magnesium. Add any other components you wish.”  Speaks to the things scientist do to add speed and specificity to the process.

Buffer is a more liquid version of cytoplasm. It is easier for the relevant particles to move about and to be healthy. Buffer is sort of like climate control.

Taq polymerase is by far the most interesting thing about PCR and the most original most mentally innovative element of PCR. Like the DNA polymerase builders in normal mitosis Taq polymerase is a construction machine on the molecular level. The remarkable thing is that Taq polymerase comes from (or in synthesized using the model of) the bacteria Thermus Aquaticus. T. Aquaticus is an extremophile. This bacteria lives near and on thermal vents (under water volcanoes) in extreme heat. It is the ability of Taq polymerase to thrive in and build nucleotides in such crazy (relative to normal DNA polymerase) temperatures that gives PCR its major start stop cycle boost. Every time the heat is turned up the Taq polymerase starts building and overtime the solution is allowed to cool it stops again, eliminating the need to wait for normal DNA building cycles.

Magnesium is required to maintain healthy levels of Taq polymerase. If the concentrations are too high the DNA will stabilize and not come apart and if concentrations are too low the Taq will not be active enough.

-

If you remember how PCR works this is not terribly complicated. RtPCR is PCR with an extra step. As you recall in PCR a sample is heated up and cooled down many times (cycled). The same thing happens here. In PCR 2 primers are needed, these mark the beginning and end of each desired segment. In RtPCR the same two primers are used but this time a probe is added. The probe looks and acts a bit like a primer. It bonds to the DNA strand in the same way, by lining up the ATGC bases. The difference is that a probe is meant to be destroyed during each cycle.

Each probe has a pair of florescent reactive particles (this is the same kind of florescent light you see in offices or in TV police procedurals). The first part of the pair is florescent light emitting and the second part is florescent light absorbing. Think of it like a star and a black hole right next to each other. The black hole absorbs all the light from the star so that it can’t be seen.

Some PCR machines have florescent light emitters and detectors in addition to their thermo cyclers (that is the part that heats up and cools down in each cycle)

During DNA division and replication the primers at the start and finish stay right where they are but the probes are in the way of the normal polymerase building action, so the polymerase just breaks them up and shoves them out of the way. The breaking up of the probe allows half of it to become florescent light emitting without interference. The black hole in this case, is far away enough from that star that the star can shine brightly.

This light is of course very very small. It takes many cycles before there is enough light to actually be seen.

The point at which there is enough light to first be detected is called the threshold cycle.

Threshold cycles are very predictable. If you start with 1 DNA copy it will take about 40 cycles to get enough copies to be detectable. If you start with 32 copies it will take 35 cycles. If you start with 1024 copies it will take 20 cycles.

There is a chart in each of the videos below to illustrate this threshold point.

Using this method you can get a very good estimate of the number of copies you have initially or “how much DNA has been extracted’.

A standard curve is used for a control in a RtPCR run. The stand curve is made up of a known concentration of a known DNA sample. If your standard curve comes out looking like you expect it to then you know your process is working the way you want it to.

It is possible for machines to malfunction or for improper handling of samples to destroy their contents. Only by always including a standard curve can you reassure yourself as to the viability of your RtPCR results.

I have two videos here that will probably be either helpful or entertaining for anyone interested in seeing RtPCR explained through animation.

https://www.youtube.com/watch?v=kvQWKcMdyS4

This guy speaks very slowly. His English is good but accented. The slowness may be a little boring but his explinations and animations are great for the basics of how RtPCR works

https://www.youtube.com/watch?v=fkUDu042xic

This guy is a very animated presenter and a little technical but if you understand PCR it is a fun presentation.