3 replies to this topic

[sl17]

I recently had some problem expressing a mutant protein (just a point mutation), while expressing and purifying wild-type is not a problem at all (if anyone has suggestions here, please share!).  Therefore, I just bought some BL21-CodonPlus(DE3)-RIPL cells, as the person who gave me the plasmid suggested (I don't know why they didn't use Rosetta or Rosetta 2 though...).  Since the cells are not cheap, I wonder

1.  Can I just follow protocols online to make my own chemically competent cells? Besides the additional plasmid for rare tRNA, these cells are not much different from other competent cells, right?

2.  I read the transformation protocol provided by the Strategene, there are two specific things required for the transformation.  The XL10-Gold
β-mercaptoethanol and 14-ml BD Falcon Polypropylene Round-Bottom Tubes.  They say the efficiency is not guaranteed if using other 2-ME or other tubes.  Has anyone used just normal 2-ME or other shapes of polypropylene tubes before?  Maybe can share how long the heat pulse should be?  I asked this because, if I want to make my own cells, and I don't have the XL10-Gold 2-ME (they don't sell it separately, either).  Also, 14ml BD Falcon tubes are not cheap.  

Thanks.

Edited by sl17, 08 August 2012 - 12:27 PM.

[bob1]

YOu can certainly mae your own competent cells, but it may depend on the plasmids that are contained in the bigs as to how propagatable they are.

So long as the B-ME is fresh it should be fine.  The round bottom tubes are what Hanahan used in his original paper describing how to make competent cells, the use has been propagated from there.  I personally use 1.5 ml eppendorf tubes and a time of 1 min.

[sl17]

Thanks!  We used the eppendorf tubes for the Bl21(DE3)pLysS we made as well.  It's good to know the source of that.  

For making own competent cells, has anyone tried to on these CodonPlus cells?  Thanks.

[bob1]

I've never used codonplus cells, so I can't comment.  However, some bugs come with a non-propagatable plasmids (I guess no origin of replication) apparently.