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.

ramping up

I have begun to run RT2 and methylation plates.

I am taking a lot of notes.

I am staring at a lot of spreadsheets.

I am aliquoting in a tiny hood.

I thought the aliquoting would be the easy bit, as I do a great deal of it already. Silly me.

Different styles of pipetters make for novice like aloquoting. That us discouraging.

My brain is a bit mushy but I am really encouraged overall.

I haven't been able to stick around for analysis yet because we are busy at my (real?other?normal?) job.

Yuye said my curves look pretty good though.

I am happy to hear it.

My forensics class is doing RtPCR and DNA amplification this week.

It is so strange to be actually studying something so immediately useful.

I don't think that has ever happened to me in a school setting.

I am, however; feeling a bit swamped. Some big projects in my regular job plus a final power point and paper project for forensics plus all the new stuff for the co-op class plus a friend coming in from Germany to stay with us this weekend plus a birthday party...

gracious!

I must admit I am really enjoying all of this.

:)

A breakthrough

So the last time I watched the PCR process the whole way through the thing that baffled me the most was the 'paperwork'.
As I am training with QC there is, of course, a great deal of documentation . . . a great deal.
This documentation takes the form of many many excel sheets. Most labs that I have been in use excel a great deal so it wasn't their spreadsheetness that was getting to me.
I was presented with a lot of names (gene names and batch names and plate names) and numbers that I absolutely didn't recognize and became quickly disoriented. It was discouraging.
Today because my main robot was down for maintenance I got a chance to see the whole process again.
This time something clicked.
I attribute this breakthrough in large part to my forensics class. We are currently studying DNA analysis and have hit the PCR section.
The textbook is not enjoyable reading so after I look it over I tend to go to YouTube and watch videos about anything that is confusing me.
I felt a lot of personal pressure this week to write good conference posts because this is my thing.
I work with RNA every day and am learning PCR right now. I have access to as many experts as I want and have the advantage of having in the concrete something the others only have in the abstract.
So I have been watching about 2 hours a day of YouTube videos on DNA replication and on PCR.
I have been really gratified with my classmates responses to my posts. Its hard to gauge how much of the positive response is the 'required number of posts to a classmate a week' and how much is genuine appreciation for my breaking things down and linking helpful videos but so far so good.
Because of all of this I went into today's PCR training with a much better idea of what I was seeing. Additionally several of the acronyms started to match up with things I already know.
When I used to make genome plates I learned Hs for Human MM for mouse Rn for Rat etc. and today I saw that in that huge list of confusing names those abbreviations featured prominently.
PPHsxxxx is a primer for human DNA PPMmxxx is for mouse and so on. I got that all the genomic DNA starts with a GH and all the cDNA starts with a "c". As a huge bonus I remembered that in China dates are written differently than they are here and suddenly the huge random string of numbers were dates 20130912 was on everything from today.
The reading breakthrough combined with better base knowledge made me feel so much better about learning this stuff.
Next Monday I will run my first RT2 plate.

independence of sorts

Yesterday, while passing in the hall, Yuye asked if I could make him some gel plates sometime before 1:30.

I was waiting on the manifold vacuum so I went into the gel room and made some.

I was worried about the lack of supervision but reassured myself that this is a very simple recipe.

I took out the finished plate from the hood and made 4 more.

I kept rushing back to make sure the microwave wasn't blowing up.

No bangs.

All is well.

At 4:30 he asked me to follow him into the room and make some plates. I told him that they were already in the fridge. He had a look and was very pleased to see them there all wrapped up and ready to go.

So apparently the unsupervised bit was my misunderstanding but we are both happy that I did it on my own.

I think I am going to just make plates every few days and keep the stock up. It was my original plan and it seems well received. 

Yay for small victories.

Explain it to me like I'm 50

Explain it to me like I'm 50...
This is what my forensics teach asked us to do: to explain DNA analysis to a 50 year old with a 5th grade education.
It was a good time for the question. All this labor day weekend, while on our anniversary trip, I talked through running gels with my husband.
He is not 50 but he is an IT guy not a biologist.
This is how I answered.

Imagine that you are standing on an unpaved road near a beach. The road is mostly sand and seashells.

Imagine you are standing on an unpaved road near a quarry. The road is mostly gavel and clay.

Imagine you are standing on an unpaved road in the mountings. The road is mostly earth and sticks with a few rocks in it.

  These roads although they serve the same purpose and may even be the same size are very different. They each contain particle elements that are quite distinct from each other. They are all influenced by their environments, by the traffic they experience and by the weather they are exposed to.

 These elements represent the DNA of the roads.

 Each person is made up of small parts that are as unique as the road components above.

DNA analysis is the process by which these parts are identified and presented.

 The process of analysis takes place using gel electrophoresis.

The gel part is very much like clear Jello.

 It looks like a flat wallet sized and shaped ‘plate’. Each gel plate has some very small wells in it, near the top in the middle and near the bottom. DNA samples with some dye in them, to make them more visible, are put into the wells.

 The electrophoresis part is a small container, just big enough to hold the plate and filled with buffer (basically salt water). The container has terminals just like a car battery. When the DNA loaded gel is in the container. The terminals are hooked up and electricity is applied.

 The electric current in the buffer is enough to push the DNA from its wells through the gel. The current is mild and only going in one direction. The bigger heavy proteins (like the rocks and shells above) move very slowly. The smaller and lighter proteins (like the sand or clay) move more quickly. It is easier for small things to move through Jello than it is for large things to move through Jello.

 After about 20 minutes the electricity is turned off and the gel is taken out to be photographed and/or analyzed.

 Each person has a different amount of big and small bits to their DNA, just as each road has different amounts of big and small bit.

 Each persons gel will necessarily display their unique make up of big and small bits and it is the job of scientist analyzing their DNA to produce a picture (literally) of what that looks like.

 I am attaching a picture of some gel plates that I made last week so you can see what they look like. The white things are molds for the wells as these gels are not set yet.

 Also NOAA has a nice picture of the DNA sample being loaded into a gel here.

http://oceanexplorer.noaa.gov/explorations/03bio/background/molecular/media/gel_plate.html

I should have said something about amplification.

I should have said something about sample size and condition and source.

I should have said something about purification too but overall I loved the question.

I was told in a math class once that if I couldn't explain something clearly that I really didn't understand it well enough.

I think that is not true for some people.

It might very well be true for me.

I am going to try to apply the question or at least the principle of the question to what I am learning here.

That should assure my assessment of my own comprehension at least a bit. 

October AAAA

So Yuye, like many of us, has maxed out his vacation time. He wont get reimbursed for most of it at the end of the year and has decided that my training can help him get in his trip to China for vacation for 4 or 5 weeks starting in October.
Yikes!
Also I really like him and think he deserves a vacation.
He says that I should be able to take over running the RT2 primers and the gels by then.
He wants to focus on RT2 because it is the most complicated thing that I will learn. He feels that no one has time to take over this for him and that once I can do RT2 everything else should be a breeze.

This helps me narrow down some timelines for Co-Op scheduling.

Wish me luck



image from http://www.qiagen.com/Products/Catalog/Assay-Technologies/Real-Time-PCR-and-RT-PCR-Reagents/~/media/NextQ/Image%20Library/S/23/45/S_2345_GEF_RT2Profiler_s/1_8.ashx?la=en

That was then

I was pretty unfocused coming out of high school. I had terrible grades despite my good schools and even though I have always loved learning I just never cared about grades or homework. It was a source of frustration for my teachers and parents but I was happy just to learn the things that interested me and not do much else. I didn't really do home work but I tested well, I didn't take part in school activities except for the biology club but I excelled in the labs. Because of these factors I had zero expectation of going into science.
I thought I might join the Navy or find some job on cruise ships or in the library or the forest service.
I have always wanted to do everything and never been particularly good and whittling that down.
Unexpectedly and blessedly my best friend decided to send me to college.
We had both been working since our sophomore years of high school and saved enough to (I thought) get an apartment near the University of Maryland where she was to attend. Instead of doing that however we wound up at her parents place and all the money went to sending me to Montgomery college.
I loved college. My grades went from D's and F's to A's and B's. I'd never had A's in my entire life. At first I assumed that this was a result of Public School being easier than Private School but eventually I came to the conclusion that being allowed to study things that drive me makes a big difference.
I studied Theater and Medicine and English and History and even to my shock finally started to get Math.
I am terrible at math. I decided to try to get algebra. I took it 8 times at Montgomery before it clicked, but when it finally clicked I zoomed right up through Trig and and Pre-Calc. I shuttered to a halt there but eventually I will get Calculus too.
All the time I was there I still had that focus problem. One day, nearly on a whim, I found myself in the Co-op office. Someone in one of my classes had recommended it as a good way to find a job. I had been working as a farm hand and a janitor and for a short while, while my dad was in rehab, helping to run my fathers landscaping business. I was ready for something less stinky and exhausting.
The woman at the Co-op office asked me what I was interested in and I said Biology, this mostly due to the fact that I had just gotten out of a great anatomy lecture...if she had asked me the day before I would have said Psychology for the same reason.
She pulled a sheet out of a drawer and asked me if I could get to Rockville and did I have a car.
I could and I did.
I went up to Molecular Medicine and interviewed for a job I knew nothing about, in a lab I had never imagined on a lucky day.
I was accepted as a student lab aid and began working part time in their dark room developing pictures of chromosomes from amniocentesis and leukemia patients. It was fascinating!
When the semester was over I stayed on and began doing other things. I washed dishes and did filing, I learned medical billing and how to argue with insurance companies and eventually I grandfathered my way into doing real lab work. I learned tissue culture and microscopy. I did karyotypes and I loved it.

from http://www.biotechnologyonline.gov.au/popups/img_karyotype.html

The lab was tiny. At most there were 8 of us there sometimes as few as 5. I worked there for many years and would be there still if we hand't been bought out by Quest Diagnostics. They offered us jobs in VA but the drive was too much.
I had finished my Associates in Biology by this point and moved onto the University of Maryland and I was about to be married.
At this point I took a job with the lab that came in to by all our equipment. After a year there they were bought out by Labcorp. I could keep the job and move to NC or find something else.
I did a year of temp work that was horrible (for Fisher/Mckesson (high stress military contracting and strange gross AARP contracting) and then landed at Qiagen.
 Qiagen was so different. I knew nothing about gene silencing but was offered a job as a genetic librarian. It sounded interesting and I was given the tour on the day after Halloween. The whole building was done up in orange and black and people were laughing and there were pumpkins all over the place.
I am sorry to say I took the job for the pumpkins.

Here are my husband and myself being the Zombies part of Plants vs Zombies at last years Qiagen Halloween Party.

I became not a genetic librarian (I do some of that of course) but instead a robot wrangler.
So much of what I do for work is automated and when I am not dressed like a white ninja in a clean room I am up to my elbows in some crazy robot trying to get it to behave.
Here I am doing my thing at 35 sec in (George is my lab mate and one time trainee his sweet son Mervyn got the interview).

I really enjoy the scripting and the never-bored quality of being a babysitter for  robots but I wish I were a tech again. I don't want to be a Production Associate. I make more money than a lot of the techs but I am not proud of my job.
I am really hoping that what I am learning for this class and for my boss will get me on a track that I am proud of.
I know what they want here at work. They want more back up for QC and they want to move someone out of my area because grants are down so academic orders are down and NIH orders are down and bla bla bla money. They want to do something kind for me as well. They want to help me do more than hang out as a peon. They have offered me management positions a number of times but I just don't want it.
I don't want meetings and I don't want to be the hire/fire person. I don't want to be a boss. I want to do things that are interesting. It still boils down to that. As a gene silencer I may have a small hand in curing cancer. I may help if even in only a minuscule way in the upcoming personal medicine revolution.
I think this class will move me closer to the degree that will let HR keep me on the science side of things and the cross-training that the class represents will get me enough real science to belong there.
I know what the school wants too.I have seen the degree completion stats. I know the workplace credit program is one of the ways schools that cater to returning students are  examining. The education market is in major flux and this sort of program is one of the directions it might pivot to (one of a slew). My success in the program (any students success) boosts those numbers and even my (or anyone's) failure helps guide the evolution of the system.
I don't understand so many things about what I am about to learn. I picked  my paper and power point topics based on the things I know the least. These are the things I feel flying over my head in the lunch room. I am hoping the class structure will force me to look closely at the things I can get away with ignoring. This is a lot about self improvement. I felt too shy to ask many of the questions that I have. I was afraid of wasting peoples time and I was afraid of looking stupid, but now I must ask.
I feel so lucky to have gotten into the program.

Here we go Co-op

This is a blog about my COOP 486 experience. I am writing it for myself and also for Dr. McLaughlin so that she can keep easy tabs on what I am up to.
I applied for the CO-OP program for a few reasons. The first reason was that I had a really good experience with the CO-OP program at Montgomery college many years ago. Additionally I knew that I had some major cross-training up-coming and thought it would be a good time to try the workplace learning for credit program out. The third reason is that I am dragging my feet on some of the upper level biology classes because I am honestly afraid I wont be able to pass them. I know I can do my work so this seemed like a good option for the upper level credits.

I was very confused at the beginning of the application process. It took a long long time (months) to hear back from anyone at UMUC about the status of my application. When I did this before it was very fast and informal and I was working for the program the same week I applied for it.
Partially this is because the program was (apparently) full for the summer semester when I applied and my application was pushed back to the fall. Partially it was because I had horrible luck getting a hold of anyone in teh work place study office. It was like one sided phone tag. lol
The other problem was that my boss wanted me to start cross-training immediately and I had to stall until the fall semester to get things lined up right.
Strangely helpful in all of this was the fact that I had some semi to very serious medical things pop-up during the summer so that draggng my feet became quite legitimate.

Now we are in week 2 of the semester and things seem to be squared around.
Last week I was more than a little worried about how things would go. Dr. McLaughlin didn't get my learning contract and Yuye (my trainer from QC) was so swamped that he had no time to train me.
I emailed Dr. McLaughlin about it and she reassured me quite a bit by telling me that things frequently start slowly but that they tend to pick up steam later on.
I am happy to report that my daily bugging of Yuye paid off yesterday and we set a firm appointment for today after lunch.
Huzzah!
I had been walked through the full QC PCR process at the beginning of the summer and the amount of detail seemed really daunting, so yesterday I asked if he would show me how to make the gel plates.

I hoped that this simple task would both help QC, as I can stock inventory fairly usefully, and give me a good foot in the door.
Nothing succeeds like success eh?
Today I watched the data analysis for several types of PCR plates and then I watched Yuye make and run a set of gels.
He then asked me if i was ready to make a few myself. I said yes and here they are!

They look OK to me. We shall see. If tomorrow no one complains then I will have the accomplishment to go with my current feeling of triumph.

Yay learning new stuff.