(Yeah, I know, luck ... at least I feel better after staying in the lab until 9:00PM purifying DNA from 40 mini-preps and then preparing 40 DNA digestion reactions.)
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Alex Palazzo is an Assistant Professor in the Department of Biochemistry at The University of Toronto.
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« Science = Faith???? | Main | Different ribosomes fulfill different functions »
This is why you choose 20 colonies ...
Category: Lab Life
Posted on: November 27, 2007 3:08 PM, by Alex Palazzo
Comments
40? I only count 20.
Posted by: Acme Scientist | November 27, 2007 4:52 PM
As I keep telling my students,
"You only need one"
Posted by: BK | November 27, 2007 5:35 PM
I needed to make 2 new constructs. I picked 20 colonies from each ligation. From the first reaction, 1/20 colonies had the insert (above figure). From the second ligation the success rate was 3/20 (data not shown). Now hopefully their sequence will be as expected.
Posted by: apalazzo | November 27, 2007 5:50 PM
I was going to say, "But will the sequence be right?" But I see you have already addressed that possibility....
Posted by: Mrs Whatsit | November 27, 2007 6:57 PM
No, that's why you need to clean up your fragments better. ;)
If you have to choose more than a couple for a cloning, you're doing something wrong (incomplete digestion, incomplete dephosphorylation, etc).
I'm going to guess your insert was a PCR product that was not gel purified so you ended up cloning a bunch of primer-dimer fragments?
At least you got it. :D
Posted by: Eric | November 27, 2007 11:35 PM
Yeah, been there.
Ever considered another brand of DNA ladder? Seriously....
PBC.
Posted by: Protobiochemist | November 28, 2007 12:55 AM
For throughput, how about crack screening? When I had a rare insert - like with hairpin siRNA cloning - I would just crack about 100 colonies.
You grow a colony in 100ul LB in a 96 well plate (and touch the tip after innoculation to a reference LB plate) for about 3 hours, you'll have a nice turbid solution. Add 2x crack, wait five minutes, and load onto a few gels.
After that, you grow the small number showing increased insert size. You save money and time. Fewer minipreps etc.
Alternatively, PCR screening can be done with most cloning constructs. Just use T3 and T7 as your PCR primers and do colony screening. Just touch the tip from a colony into a PCR mix (don't let it sit too long before running), cook for at least 5 minutes to lyse the bugs, run 40 cycles, then look for long inserts. A annealing temp of 55 works fine.
Posted by: MarkH | November 28, 2007 1:16 AM
MarkH: What is 2x crack? It sounds like something you buy off the street corner.... I tried googling crack sceening. I got some very strange hits.
Posted by: Mrs Whatsit | November 28, 2007 9:03 AM
Crack buffer?
100 mM NaOH, 10 mM EDTA pH 8.0, 1% SDS, 10% glycerol, and your favorite gel-running dye. I add a little bromphenol blue, the green band always seems to land right on top of my bands and pisses me off.
Basically it's just SDS lysis buffer for the bacteria that prevents rapid DNA degradation (EDTA helps) and thickens the solution for running on a gel.
Posted by: MarkH | November 29, 2007 12:46 AM
And they say that there's no science on scienceblogs.
Posted by: apalazzo | November 29, 2007 8:00 AM
Colony PCR is the only way to go... except when it doesn't work.
Posted by: Nick Anthis | November 29, 2007 9:47 AM
No, that's why you need to clean up your fragments better. ;)
Or, you just need to get better at choosing which colonies to pick. They're not all the same - if you pay attention, and with a little experience, you can get better at spotting the positives. Like I tell my students, it's all about knowing which ones to pick.
Posted by: bayman | December 1, 2007 2:07 AM
Getting the right clone is one of the most satisfying experiences in biomedical research. This is because it is pure unadulterated pleasure: no question of interpretation, no question of significance. It's just wanting something very specific, and getting exactly that, period.
One funny experience I had as a post-doc was with a non-directional ligation. Since my +insert transformation yielded hundreds of colonies more than -insert negative control, I only picked 8 colonies. All eight had the insert in the correct orientation. On my way home I bought a lottery ticket.
Posted by: PhysioProf | December 1, 2007 6:01 PM
"They're not all the same - if you pay attention, and with a little experience, you can get better at spotting the positives."
Yeah. Sometimes the positives have a slightly different translucence or size than the recircularized negatives, especially with large inserts. It's been years since of done any subcloning with my own hands, but this discussion is whetting my appetite. If only I had the time. Of all of the huge number of methodologies I have mastered in the course of my scientific career, the most fun for me is molecular cloning, and by a large margin.
Posted by: PhysioProf | December 1, 2007 6:04 PM
This was the only simple info on crack-screening I could find on the web!!! So, thanks MarkH!!
Posted by: Nick | November 6, 2009 5:23 AM