MidReal Story
Gene Editing Chronicles: PCR Breakthrough
Scenario: l'm a third-year PhD candidate in biology, coming from China, specializing in gene editing science. My daily experiment is PCR and cell culture.
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I have a problem, and I’m not sure what to do about it.
The problem is that I’m too good at my job.
It’s not that I have a huge ego or anything, but it’s hard to deny.
And after all, I’ve been doing this for three years.
I’ve gotten very good at it.
So good that I don’t even need to think about it anymore.
I just go through the motions and the experiments are done and the work is finished.
And then I get sad, because it’s over and there is nothing else to do but wait for the next day.
That’s how it goes every day in our lab.
We are all very dedicated here.
We have to be, because our work is very important, and if we don’t do it correctly, everything will be lost.
We can’t make mistakes.
We have to be very thorough, precise, and organized.
We have to collect all the data and analyze it carefully.
We have to make sure that we are correct in our conclusions.
It’s not easy.
But I do my part, of course.
I’m a third-year PhD candidate in biology, studying gene editing.
I’m from China, so I came a long way to be here.
But I’m very happy to be here, because our lab is one of the best in the world for this kind of research.
I feel very lucky to be able to work here with Professor Johnson.
He is a genius, and he is always coming up with new ideas and new techniques for us to try.
Right now we are testing a new PCR method that he developed himself, and he says that it will give us much better results than anything else we’ve ever used before.
That’s a big deal, because most of what we do in the lab revolves around PCR and cell culture.
It’s not particularly exciting work, but it’s important because we need to be able to genetically modify cells in order to study them properly.
This morning I got up early as usual so that I could prepare solutions and media for cell culture before anyone else got here.
The cells we use are very delicate, so you have to be careful with them.
Even though they are only lines of cells growing in dishes, they are still living organisms, and you have to treat them with respect and care if you want them to grow properly for you so you can study them later on.
gene editing
After I finished the media and put it in the incubator to warm up to the proper temperature, I went back to the lab to start my PCR experiments.
A lot of people think that PCR is boring and repetitive, but I like it because it’s very precise and orderly.
You have to be very careful about everything you do when you’re doing PCR, because even the smallest mistake or contamination can ruin your experiment.
So you have to make sure that you are very organized and methodical in your approach.
If you are not organized or if you try to take shortcuts, you will pay for it later.
And we don’t want that.
The first step in the process is to make a master mix of reagents in a sterile tube.
You will need to add water, Taq polymerase, your primers, buffer, and dNTPs.
It’s very important to make sure that you add all the reagents in the correct order and that you mix everything well.
Then you need to put your tubes in a rack next to your DNA samples so you can add them quickly and accurately without risk of contamination.
When you’re ready to add your DNA samples, first make sure that all the tubes are closed tightly so nothing will spill out.
Then you can use the pipette to add a small amount of DNA to each tube.
It’s important to always change your pipette tips between samples to avoid contamination.
After you’ve added the DNA samples, give the tubes a gentle shake to mix the contents and put them in the thermal cycler.
Make sure that you seal your tubes with parafilm or tape so that they won’t evaporate during the reaction.
When you’re ready to start the machine, make sure you select the correct program for your experiment and then close the lid securely.
You will hear a little beep sound as the machine begins to heat up for its first cycle.
That’s when you know it’s working and everything is going as planned.
PCR has been an important part of my research for many years.
It’s a very useful technique for amplifying specific DNA sequences and detecting gene expression patterns.
And it’s a good thing, too, because I’ve been doing the same PCR experiment over and over again for the past six months with nothing to show for it.
I’m not sure what’s going on, because I’ve followed the protocol exactly as it’s written, and I made sure to use fresh reagents and to avoid contamination at all costs.
But no matter what I do, I can never get my target gene to amplify properly.
I don’t know why this is happening, or what else I can do about it, but it’s very frustrating to spend so much time working on something only to fail repeatedly with no explanation as to why.
I even wrote an email to Professor Johnson last night to explain the problems I’m having with the PCR method he gave me, and I asked him if he could suggest anything else I could try to get better results.
I made sure to include all the details of the experiments I’d done so far, along with images of my gel results showing no amplification of my target gene.
I am a boy
As usual, I get started on my PCR experiments early in the morning before anyone else arrives at the lab, because they take a long time to run and I have other things to do later in the day, like cell culture and preparing solutions for everyone else’s experiments.
There are several problems with doing this, of course, including the fact that I am not a morning person and it is hard for me to drag myself out of bed when the sun isn’t even up yet.
But at least this way I can finish my PCR experiments sooner than usual (since they always fail) and have more time to work on other things later in the day (like troubleshooting why they didn’t work).
So I try to get here as early as possible every morning to set up my reactions while avoiding the rush hour traffic on the subway, and still have enough time left over to get a cup of coffee before I start working for the day.
After mixing together my DNA samples with the master mix reagents, I start checking my notes to make sure I haven’t forgotten anything before I put the tubes into the thermal cycler.
I review the protocol one more time to verify the correct settings, the proper volume of reagents to use, and any other important details I should be aware of.
Everything looks good to me, so I close my notebook and move the tubes to the machine.
As soon as I put them inside, however, I realize there is a serious problem: there is no amplification control!
I must have forgotten to add it to the reaction mix along with my samples.
I curse myself silently for making such a careless mistake, because now there is no way to know if any of my samples amplified successfully or not.
If they don’t amplify, then all my samples are useless, because there will be no other way to tell which ones contain the target gene edited by CRISPR/Cas9 technology.
This means that six months of work (and several hundred dollars) will have been wasted on a failed experiment, and I will have no results to show Professor Johnson when he asks for them.
I’m starting to panic now, because I don’t know what to do about this problem.
How could I have overlooked such an important step when setting up my PCR reactions?
As I sit there, staring blankly at the thermal cycler, I suddenly remember another boy who was working in our lab a few years ago, when I first came here for my Master’s degree.
His name was Li Wei, and he was from China, like me, and we were both studying molecular biology with Professor Johnson at the time.
Li Wei was very detail-oriented and analytical, and he always made sure to check everything carefully before starting an experiment, which is why our project was able to continue running after we lost power during a hurricane and had to wait two weeks for it to be restored.
He even managed to find a way to improvise a thermal cycler from a rice cooker by using a water bath, so we could finish our PCR experiments and get our results on time.
If not for his quick thinking and ingenuity, we might have lost all of our samples (and our funding) and been forced to start all over again from scratch.
As I remember this, I am struck by a sudden idea: what if I use a gradient PCR instead of a regular one?
Our current method of detecting gene editing is not working very well, because it misses a lot of edited genes due to low efficiency.
I know that Professor Johnson is already planning to switch to a new detection method called next-generation sequencing, but this will not be available for at least another year or two.
So I decide to modify the protocol myself and see if I can increase our detection rate by at least ten times, or even more!
What do you think of this?
I’m sorry, Li Wei, but I don’t have time for this right now.
I’m trying to set up a PCR experiment before everyone else gets here and I haven’t even added my samples yet.
Can you just take a look at it and tell me if it looks okay?
Li Wei comes over and stands beside me at the bench, looking down at my notebook and checking all my notes carefully.
He is wearing his glasses today and has his hair tied back in a ponytail, so he looks even smarter than usual.
This makes me feel a little bit self-conscious and nervous, because I don’t know what he is thinking about my experiment and whether I have made any mistakes.
But he doesn’t say anything or make any comments about it, so I guess it looks okay to him.
Or maybe he just doesn’t want to embarrass me in front of everyone else by pointing out any errors I might have made.
After we finish setting up the experiment, Li Wei goes back to his bench to work on his own project, while I put my tubes into the thermal cycler and start running them at 95°C.
Half an hour later, when they are finally finished, I take them out of the machine and check them carefully under UV light to see if any of my samples amplified or not.
As soon as I look at the first tube, I am shocked to see something very strange: There is more than one edited gene in it!
I’m not sure how this is possible, because we only edited one gene with CRISPR/Cas9 technology in our cell culture experiments, but it is definitely there in my results, along with another one that I can’t identify by name, because it has no location or description in our list of genes or proteins and no known function that I can find in any of our databases.
I check all my other samples, as well as the negative control (which shows no amplification), and they all have edited genes in them too!
I can’t believe what I’m seeing right now, because none of these edited genes were visible in my previous experiments, which means my new PCR protocol is working better than expected!
This is amazing!
I have to tell Professor Johnson about it right away, so he can come down here and take a look for himself!
I take out my phone and call him on his cell phone number, but he doesn’t answer it right away because he is probably busy working on his own project in his office.
So I leave a message for him to call me back when he gets a chance and go back to my bench to check with Li Wei to see if he has finished with his experiment yet.
As soon as he sees me coming over, Li Wei puts down his pipette and comes over to meet me at the bench.
I don’t know if I should be happy or angry that you are interrupting me while I am working, because you know how much I hate it when people do that to me,” he says in a serious tone of voice that makes me feel a little bit nervous and unsure of myself.
But I am also very curious to know what you have discovered from this experiment and whether you have made any progress with it, so I came over here as soon as I could to find out for myself.
