My friends never understand what it is I do all day in the lab.
So, I thought I’d tell you.
Last week I started a project to clone three genes, the first step being an amplification of DNA (specifically, the genes) via a technique known as “PCR.”
PCR was invented by a wild man, Kary Mullis.
(you must check out this guy, his reading list and autobiography!)
For his efforts he won a Nobel Prize. But now, PCR is basic molecular biology.
And last week I could not get my PCR to work.
I went home Friday evening pretty frustrated and Monday morning found me pouring over technical literature to formulate a plan.
I also did an online search which totally distracted me with laughter.
I have cut and pasted the online chatter down below.
I find these unknown colleagues both heart warming and a perfect reflection.
This is What I Do.
This is Science as I have come to understand its practice in molecular genetics.
Ninety-percent of what we try to do fails.
We follow the best logic we can muster…
and in the end often conclude, “it was my lucky red t-shirt,” - i.e. we do not know.
Perhaps, that’s why I love this painting I post here of the red chair, by Karen Cleveland.
Karen paints with her energy and intuition and her work to me embodies consciousness.
Science that works has this same wild artfulness.
The sky around that Red Chair could be the stuff percolating in my PCR.
And now, my virtual colleagues, and their artfulness which runs like this...
Posted by: vetticus
Hi,
I've said this before, but, basically, i've been using a PCR reaction with Pfu on genomic DNA, and until very recently, it's worked really well. last few weeks, the reaction flopped majorly. Other researchers in our lab have used the Pfu also, unsuccessfully.
So, we got a new batch out of our freezer, it too didn't work.
Changed the water... didn't work.
Fresh dNTP... didn't work.
Fresh primers... didn't work.
Changed the PCR machine from my floor to the one upstairs... didn't work.
swore... didn't work.
changed from Pfu to another proofreader with it's very own buffer and with assurances that this PCR would work... didn't work
(though did get some lovely primer dimers)....
I was wondering... and was hoping someone could confirm this.... the only thing that has remained constant is my supply of DMSO (5%). could this be the thorn that screwing me around?
(as i write this, i'm running a PCR with a fresh batch of DMSO)
can DMSO go bad?
hoping desperately that this reaction works.
supposing this reaction doesn't work, and i manage not to throw myself off a building, is there anything else i can try?
this reaction has worked for months, and now is not.
vetticus
PS
fresh DMSO... didn't work. i don't get it. I got this reaction to work. One day it's fine... next it's stuffed. Which molecular biology god should i make a sacrifice to?
Posted by: Bob
Hi,
I am fairly sure that DMSO degrades slowly in the presence of water, it could be that the
5% solution you are using is degraded and seeing your last comment, it could be that the new stock is also degraded if stored as 5%.
Just a thought- try fresh DNA, if possible, it is barely likely but it could be the factor as it seems to be the only one not changed.
Otherwise I am flummoxed!
Good luck
Bob
Posted by: vetticus
apparently DMSO degrades if exposed to sunlight... ahhhh.
still doesn't help me out (but useful for anyone else out there). Don’t store it at 5%, that's the percentage of final volume...
my head hurts.
trying fresh DNA... will probably have result in ~30 min.
thinking about it all day... the only other variable that has changed is that the other lab swiped our radio and is playing pop rock instead of our usual indy/alternative.
i don't know.
perhaps i should stand on one leg whist preparing the reaction.
Post by: nabla
“the only other variable that has changed is that the other lab swiped our radio and is playing pop rock instead of our usual indy/alternative.” That is a quiet good reason according to my opinion!
Posted by: littlecell
there must have been something wrong with your DNA template.
please store the template at 4 centigrade but not freeze them.
good luck!
Posted by: vetticus
It works... it works.... it works... little dance little dance.... it works...
what happened... i offered a blood sacrifice to one of the more fickle gods of molecular biology (ok, maybe not, but the idea did cross my mind)...
seriously, i have no idea what happened.
it just stopped working, but it works now...little dance little dance.
wearing my lucky red t-shirt must have pushed it over the line.
the DNA's fine, the primers are fine, the magnesium content is fine, the water is fine, the machine is fine.... little dance little dance little dance.
what doesn't kill me can only make me stronger.
can finally breath normally again.
Posted by: robyn
Hi guys,
I am having the same problem!
2 months ago It worked OK.
A colleague suggested that it would work better at Ta=67. I tried that - it worked beautifully!!!
Now, suddenly at Ta=67 there is nothing!
I know what you're thinking… Not so.
I tested it again at 67 - no luck.
Tested it with new primers and new dNTPs and new water - no luck.
Decided to play around with the temperature - tried 65 (so-so), 68 (nothing), 62 (so-so)…
Any help will be greatly appreciated as I don’t have any more hair left to pull out!!
Posted by: perneseblue
"what doesn't kill me can only make me stronger.”
Except a horrible disfiguring accident that leaves you paralyzed for life
And what was it, finally? The DMSO?
P. Bralley (not posted there but here)…
I decreased my DNA and enzyme, increased the magnesium, dropped the DMSO completely.
It worked! …“little dance little dance”
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