2 replies to this topic

[lschier]

Hi guys,
Is the first time that I’m working with competence, and I’m  having a huge problem about how to make competent cells. I’m working with XL1-blue strain, and I need at least 10^8 for testing my cDNA library. But the problem is: my best result was just 7.7 x 10^7 per ug. I tried many protocols, but I had only success in Nishimura protocol adapted by Rosati (Bioruffo.net). So, I going to describe what I have done!
I grew a single colony in 2 ml of LB medium overnight. Then, I put 1 ml of a started culture into 50 ml of SOB medium (the last time that I did this protocol, I put 2 mM of MgCl2). The culture grew for 16 hours at 18*C with moderate shaker (Inoue protocol) , and then I started to check the OD (normally in the next day) until reaching 0.4. After that, I centrifugated the cells at 4*C, 10 min at 3900g. The pellet was resuspended into 6 ml of cold TSB ( LB medium pH 6.1, 10% PEG, 5% DMSO, 10mM MgCl2, 10mM MgSO4 ) and incubated on ice for 15 min. Next, I started the transformation protocol,  that are composed of 100ul of cells, 20 ul of KCM5x  (KCl  0.5 M, CaCl2  0.15 M and MgCl2  0.25 M) 1ul of DNA (0.1 pg/ul) and 79 ul of pure water. I left it on ice for 20 minutes and RT for 10 minutes (without heat shock – Nishimura protocol). I added 600ul of SOC medium, and incubated on water bath for 1h30min. Finally, I plate 20ul of cells into LB-Amp plates and transfer to the incubator at 37*C for 12-16h.
I can not do the heat shock because I don’t have the special polypropylene tubes for it, so I tried to use PCR microtubes. I counted only 10 colonies using this technique, so…  :b Forget it!
Okay, this protocol works well, but how I can improve this for reaching the 10^8 / 10^9?! I’m trying the same protocol since march!! Please, help me!

[phage434]

I honestly don't know of a single person who uses those tubes to do transformations.  It's a carry over from some ancient ritual, best totally ignored.  I use 2 ml eppendorfs.  I add cells and DNA, heat shock, and then add SOC.  The tubes are large enough and spacious enough that the transformation can be shaken directly in those tubes for an hour before plating.  The shaking works better than in 1.7 ml tubes, because the cells don't get caught in the narrow bottom.

After trying every protocol in the book, I developed the one on Openwetware, which, at least in my hands, works reliably.  I don't think it hits 10^9, but will certainly hit 10^8.  Have you thought about electroporation for making a library?

http://openwetware.o...competent_cells

[lschier]

Hi!

The biggest problem for me is the heat shock. Once, I tried to use eppendorfs tubes, but didn't work. So, I chose PCR microtubes because of the "thin wall" (to get a homogeneous heat faster) . But I'll try your protocol with 2 ml eppendorfs! You have any tip about this for me?

About the electroporation, we don't have an electroporator in our lab   Surely, is the best way!

Thanks for the help!