Growing liquid cultures in microfuge tubes?
Posted 12 March 2010 - 10:36 AM
Posted 12 March 2010 - 11:43 AM
You can buy 5 ml round-bottomed polystyrene tubes with plastic snap-caps, but they don't have sufficient depth to stay put if not snapped down all the way. The rotation of the platform will shake them off in no time.
Just grow them in proper tubes; then you'll know that you have sufficient oxygen and medium for your bugs.
edited to add: I just noticed you mentioned a water bath. Why not an incubator?
Edited by lab rat, 12 March 2010 - 11:44 AM.
Posted 12 March 2010 - 12:04 PM
I think your proposed method will lead to lots of problems, contamination, and not enough oxygen getting in!
Posted 20 March 2010 - 12:06 PM
1) PCR screening
You can screen 100 inserts with prep time in a matter of hours!!!
If you have the sequencing primers for the vector and or the primers used to clone the insert, you can run this with your regular PCR reagents.
To screen for the presence of the insert use the sequencing primers for the vector.
To screen for insert and directionality use one primer for the vector and one for the gene.
I'll attach my set-up protocol that works like a champ!
Set up each well like a regular PCR reaction.
We did 10 ul rxs
4.8 ul H20
5.0 ul Master Mix
0.1 ul Primer 1 (100 uM concentrations)
0.1 ul Primer 2 (100 uM concentrations)
1.0 ul DNA from "Cells" microplate
My second choice for screening colonies for the presence of an insert is a version of the Epilyse Kit
Here there's no cell culture, minipreps, or restriction digest.
Will only tell show you size differences of empty vector and vector with insert, not directionality.
This can be done in one day. We made our own reagents. I've since left that lab, but found a paper that gives the recipes. See below.
Here's a link to a paper that gives recipes for the two solution needed:
See page 59 Section 18.104.22.168 Method 1 Ligation and Screening
If you still want to culture the colonies and prep and cut each one, I used 1ml of media in 2ml tubes, using a rich media and shaking at 37C overnight which worked fine.
I've updated this protocol 04-19-2010