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)
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.