Trouble Shooting for Transformation - (Jul/12/2012 )
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.
Sounds like slow growth, perhaps because of the protein being made.
the efficiency of competent cells can't be deduced from these controls.
You would have to do a control transformation in order to rule out this possibility.