Finally after a month of preparatory work, I was ready for my first trial run of molecular library construction. I was pretty optimistic, because I had everything carefully planned, right down to the number of eppendorf tubes that I had to use. I even took the pain to read several different protocols and scrutinize my experiment planning from all (perceived) possible angles. The only thing I did not do was read the directions of how to use the commercial competent cells properly. When I actually did, I came out with this mind-blowing numbers:
To make 1 library, I need:
- 400 eppendorf tubes for cell transformation
- 2000 agar plates for spreading of library culture
Because I have 7 library combinations, I need:
- 2800 eppendorf tubes for cell transformation
- 14000 agar plates for spreading of library culture
Essentially, I have to transform 2800 tubes of cells and spread 14000 agar plates in one go.
Say I take 1 minute to spread one agar plate, it would take me 233 hours, which is approximately 24/7 for 10 days, to finish spreading all the plates.
-_-'''
That is if I sit at the biosafety cabinet (BSC) for 10 days straight without leaving the BSC even for a split second.
My eyes nearly popped out when I saw the ridiculous figures. Forget the BSC-thing, even if I put all the incubators in the lab together, there isn't sufficient place to incubate 14000 plates!!
Now don't even entertain the thought of me spreading 14000 plates. Anybody knows that is quite impossible to be done. However, my supervisor, mentor, colleagues and myself had a good laugh at the scale of the experiment. We then worked out an alternative strategy to do the experiment, MINUS 14000 plates. *phew...*
So, after tweaking the protocol a little, I run the trial experiment at a manageable scale. I followed the recommended protocol strictly with hopes to generate as many colonies as promised by the company protocol. When I put in the plates in for overnight incubation, I was quite optimistic and very hopeful to see a lawn of colonies when I return the next day. I mean, afterall, they did say I'm supposed to obtain millions of colonies!
I waited and waited and waited till the hour came, ran to the incubator excitedly to take out my plates. どきどき。My heartbeat increased as I anticipated the lawn of E.coli on my plates. Chuckling gleefully, I reached for the plates and turned them over for a good look....... NO COLONY! I reached for another stack of plates..... ALSO NO COLONY! My heart sank as I reached for the final 3 stacks.... SPARSE COLONIES!
"Oh nooooo, what had happened to my cells?? I have no library??!! Even with using the most competent cells?? Dieeee......," - was all that was running in my head.
I supposed research isn't without experimental failures. Now I'm tweaking my strategies again and hopefully my next round of trial will bear fruit... or rather, fat juicy colonies.
Don't tell me I really have to spread 14000 plates?....
～みはる～
By
0 penciled thoughts:
Post a Comment