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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.

I recently have some strange experience/problems when I tried to purify a protein using BL21(DE3)pLysS cells.  

My plasmid (pTYB1) has Amp resistance, and I've used it to purify proteins twice in the past month, without any problem.  However, when I tried to do the transformation and purification again last week, there was no colony at all on my plate (LB+100ug/ml amp).  I thought something's wrong with the plate, so I made a new batch of plates just yesterday, and also new Amp stock.  However, when I checked the plates today after 13 hours of incubation time, I got the following weird results:  

• Plate A: plate from new batch, BL21(DE3)pLysS with pTYB1, no colony (there should have been some!)
  
• Plate B: plate from new batch, BL21(DE3)pLysS without pTYB1, no colony (negative control)
  
• Plate C: plate from new batch, some bacteria with amp resistance, serving as positive control, lots of colonies (positive control)
  
• Plate D: plate from old batch, half with BL21(DE3)pLysS with pTYB1, half without.  The half with pTYB1 has some colonies, while the other half has no colony (expected results)

I don't understand what's wrong.  If the plates from new batch have some problems, then I should not have got the right results for the controls (Plate B and C).  If the cells have some problems, then how come I got some colonies on the old plate (Plate D).  I streaked these four plates at the same time, same conditions, so there shouldn't be other problems.

The even weirder thing is, when I checked the plate two hours after the first time I checked them, Plate (A) have some colonies, which later was confirmed that they're not contamination by growing in TB+100ug/ml amp liquid medium.  What does that mean?  

This results are really confusing.  Has anyone have the same experience before?  Thanks.

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