Monday, December 7, 2009

Cloning tard

That's me, at least for the last couple of days. Typically, I am a cloning rock star, but a combination of arrogance and laziness resulted in a 1 day process taking 3 days.

Here is the situation.
Currently, I'm in the midst of a protein trouble-shooting extravaganza and as a result my work load is significantly lighter than usual. To fill the gaps, I decided to pick up some cloning that I abandoned this summer.

2 days ago:
After much digging around in the freezer, I located the five abandoned PCR products, ligated them into a vector and transformed the ligation.
That's pretty standard. I left feeling confident that I would find five plates containing beautiful colonies the next morning.
1 day ago:
Success! The perfect amount of colonies were scattered about each plate. You know, not so many that you can 't pick a single colony, but not so few as too indicate that all you probably got back was a bunch of bullshit re-ligated vector.
Anyway, I colony PCR a few colonies off of each plate and run a gel, only to find that I didn't get a single PCR product! WTF? This doesn't least not to me. I stair at the computer screen in disbelief for a few seconds when I realize that in addition to a lack of positive results, I also lacked a single negative result. I should have one or the other.
I dig back into the recesses of my mind and vaguely remember that the inserts in this case would be pretty long. Most likely, I didn't use a long enough extension time.
I set up another colony PCR, this time with a longer extension. I run this PCR out on a gel and this time I see a couple of PCR products, but most of the gel is black. Seriously, WTF? A couple? What am I? A first year grad. student.
At this point, it's the end of the day and I really want to get the hell out of there, but I am not willing to admit defeat, so I decide to inoculate a couple of colonies from each plate.
I verify the long way (the short way being colony PCR the day after you transform). I mini-prep the overnight cultures, digest the plasmids and run a gel. The image of the digest comes up on the screen and it's all I can do not to yell out MOTHERFUCKING FUCK! It looks like all empty vector, except for the two that I already knew about from the day before. I walk down the hall in a huff toward my office.
I'm ready to trash three of the plates, but instead I decide that maybe, just maybe it's time I actually look at a gel from the summer and see exactly what damn size the PCR products I dug out of the freezer were in the first place.
Well, what do you fucking know? Three (that's right three) of the PCR products are exactly the same size as the vector I ligated them into. Damn it. The digestion was a complete waste of time.
I don't feel like digesting again with an additional enzyme because I don't know which one to use and at this point I am not in the mood to look it up (despite the fact that not looking shit up in the first place is exactly what got me into this mess). I decide once again on PCR, but this time I will actually use the correct extension time.
Conclusion, almost every vector contained the appropriate insert.

I could have found this out yesterday if I had just looked up the gel in the first fucking place and used the correct extension time for the first round of colony PCR. Now, I'm behind, I feel like an idiot and it's only Monday.


Dr. A said...

I myself abandoned some cloning this summer.. thanks for the reminder!

Don't worry, you will return to rock star status soon and will have worked out kinks so that I can email you with questions :)

quietandsmalladventures said...

hey it worked out in the end, your status is intact! i, however, seem to be cloning inept. it took 5 months to get 1 of my inserts on a shuttle vector. i am not proud of this and i still have to get it out of E. coli and into Staph. please send rockstar karma my way!!

microbiologist xx said...

Dr. A - I hope your cloning goes smoothly. Feel free to email me at micro.xx at gmail dot com.

QASA - You're right! It did work out in the end and it could have been a lot worse. I'm glad you got the ligating part done. Good luck with transitioning into Staph. Some of those clinical isolates are a bitch to electroporate. The karma is on its way. :)