How many times do I need to remake a stupid buffer before realizing that the buffer is too viscous not because I am incapable of making a 50% glycerol stock. No no, that would be too easy. It is to viscous because I am unable to calculate how many grams of NaCl I need to have 137 mM in 100ml.
That would O.8 grams not 8 grams, in case you were all wondering. Stupid freaking decimal points. That only wasted a day and half.
I then proceeded work on doing a particular IP the last time. I figured out all the issues with my western blot staining (my secondaries were no longer working). I was very diligent on the experiment.
- I calculated the protein concentration in my lysate
- I ensured that I took out 50 ug of protein to run in my control lane
- I calculated / double checked all my antibody concentration to ensure that it was all optimal for the pull down
- I ran my gel super slow to ensure nice clean bands
- I used fresh new antibodies solutions (took abs out of aliquots that I had shown work)
And if blogger was working properly right now, I would show you an lovely Licor western blog, nice sharp bands, beautiful specific binding. It would've have been a beautiful publication image.
Except for those damn bubbles in the transfer...grumble grumble grumble. Expletive, Expletive Expletive!
As a side note awesome PI thinks its hilarious and pointed out all the useful data we were able to get from the experiment. Which is positive. Then she told me how she had to redo a western 6 times to get it looking nice enough for publication....
stupid transfer...
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10 comments:
Glad I'm not alone blogging buddy. :) Also glad your Western finally worked - fuck the bubbles!
Oh God. Stoichiometry. I'm suddenly remembering Chemistry. It's all coming back to me now. I'm going back to the garden.
Gratz on getting it all to work finally!
Hates the solutions, hates them. And I do biochemistry! So, you're definitely not alone. I've been struggling with ligations-- ligations I tell you! How hard can ligating two pieces of DNA be?!?
Anyhow, I'm glad that you're western worked! I'm with AA, screw the bubbles! It worked and now you don't have to troubleshoot that any more :-)
I feel your pain! I once wasted weeks trying to do an old fashioned DNA phenol extraction determined that I was not getting a decent yield because there was much to much polysaccharide that was blocking my sucking up the DNA. Of course the viscous polysacchairde was actually just really pure DNA and I had bucket loads!!
Ooohh, you use a Licor? As frustrating as it is to get pretty blots, it could be worse: at least you are getting the result. I assume. Since you didn't complain about that.
To Amanda: Ligations are simple in theory. In reality, they suck. You know it should work: but it doesn't work. No reason. I hate em.
AA - data is data isn't it? Why is it when you really want something to look nice, is the one time you have air bubbles in the transfer??
Mrs.Spit - Good God is right! Yes why don't you go out into that garden of yours and enjoy the sun, while I sit in a room with no windows trying to understand how a 1.4Mb file magically turned into a 0.4MB file during a simple transfer from one folder to another?
Mimi- thanks!
Amanda - THank you!! I don't understand what happened to me. I used to be good in math. Give me simultaneous equations and I can solve them. Derivatives? No problem. Integrals, psh piece of cake! Dilutions? God help me!
Dr.J - that's an awesome story!
1) I gave up solution calculations a while ago
http://www.graphpad.com/quickcalcs/Molarityform.cfm
Even when the calculations are simple and basically require moving decimal places, I appreciate the peace of mind. Actually, ESPECIALLY when the calculations are simple.
2) When ligations go awry more than once or twice, it is usually the fault of the enzyme, the buffer, or both. Although people will freak out if you leave RNA or restriction enzymes out on the bench overnight, many times those will survive. Ligation enzymes and buffer? Not so much.
3) Bubbles during the transfer are always the fault of lab gremlins sensing the importance of the experiment. The only foolproof way to avoid them is to perform ritual human sacrifice before each transfer.
"redo WB for 6 times for publications" ... yeah... in the times before digital cameras i.e. my phd time.
I was ready to sacrifice anything to the gods to make it happen. After a few useless ones I fianlly got a good one and I can't tell how happy I was.
Give the next WB hell :)
Hee hee, you sound like my labbers!! I am always making them re-do their Westerns so they look cleaner. :)
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