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Trouble Shooting for Transformation

Bl21 plasmid E. coli

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#1 sl17

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Posted 12 July 2012 - 03:41 PM

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.

The

#2 phage434

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Posted 12 July 2012 - 10:14 PM

Sounds like slow growth, perhaps because of the protein being made.

#3 Kamran

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Posted 13 July 2012 - 05:19 AM

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.





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