Monday, April 7, 2014

Troubleshooting Terrors

I've been learning to transform protoplast and my first experience went smashingly! However, the next attempt had a very low survival rate (~50%) and I could not find a transformed cell! All of my solutions were freshly prepared, so it's not an age/storage issue. I used the same plasmid DNA so they should have transformed. Our microscope is behaving strangely, the computer died so I cannot use the software and have to do it by hand. This should not impact their survival rate but could be giving me false information on the transformation rate. I started another one tonight and I'm going to do a GUS assay and Western blots on the samples I collected last week + tomorrow to see if it's just the microscopy that is the issue or the actual transformation.

Even in established protocols, things go wrong. Sometimes you know what and how to fix it, sometimes its completely random. For example, a few weeks ago, I did a PCR with different primers but the same DNA as my previous one.. and got no reaction. But I realized that I had used the same protocol and not adjusted for the fact that I had different primers. With PCR, temperature is critical and every set of primers has it's own "magic" temperature. So I had screwed it up! Operator error, my favorite kind! Straightforward troubleshooting and we move on.

But then I did another PCR using the exact same DNA that I had amplified before, primers that I had used before, and our stock master mix. Did not work, no bands no smears, nothing. I scratched my head because everything had been used in previous reactions so I could not understand what had happened. I checked my protocol, the temperature was correct, the cycles were correct, everything checked out.
I tried it again and replaced the ONE thing that had not been proven in other reactions, the water. Now we use special purified/filtered water that has been autoclaved so that it should have NO contaminants in it. But it also a shared commodity so sometimes they go bad before they are gone. Boom perfection. Seriously.. water.. WATER?! This is how sensitive PCR can be.

Thankfully, I have been doing PCR's routinely since year 1 Masters, so I know how to troubleshoot and often guess correctly on what might be causing my problem, even if it is something as silly as water. These protoplasts are new to me, I'm going off of a thesis of a previous lab member and 2 published papers and while there are some helpful hints, it is far from step by step. And no current lab member has done this before, so I have no one to go ask. Even my PI is only vaguely familiar. So I get to play troubleshooting in a situation where I know very little. Which means lots of reading, lots of guessing and simply trying stuff until it works!

Troubleshooting, especially in the situation where you have NO ideas, is a great skill to learn. It is also a thing I detest! I am a control freak, I can admit it. I like to have a clue, a plan, a road map! When things do not work, that should, when my road map looks more like Daffy Duck's from Duck Dodgers and the 24th 1/2 century's map, I freak out a little bit. Ok, a lot. I actually had to just go home last week after my transformation went so awry. I could have tried to start another one immediately, but I wanted to spend the weekend trying to find a new plan. I used the time to study for quals (which are rapidly approaching *PANIC*). Over the weekend, I did read and I think I came up with a plan, but only time will tell. For now I guess I will just have to try and channel Mr. Tim Gunn and

What do you do when your road map suddenly goes from a beautiful path to insanely complicated? Does it drive you insane or are you able to adapt on the fly?

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