IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/15

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Transformation ECE188, 189, 190 into E.coli

- Results : only our control plate grew!!

- Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA.

Plasmid miniprep ECE 151 (x2), ECE 153, ECE 166

  • Nanodrop results :
260/280 ng/μL
ECE 151 (3) 1.79 86.8
ECE 151 1.88 106.3
ECE 153 1.80 32.2
ECE 166 1.87 36.2


Xylose Induction with ECE 153

- 4 Tubes

- No fluorescence! We have to think about the transformation of integration vectors into Bacillus.


Glycerol Stock Transformation

- Spin down cells (competent) from 5/8 and 13/8 stocks

- Remove liquid, resuspend in Medium B

- Incubate for 60 mins at 37°C

- Add ECE 166 into tubes

- Incubate for 30 mins at 37°C

- Plate out on CM 5 plates

- 4 plates

  • 771 (5/8) + ECE 166
  • 771 (5/8) Control
  • 771 (13/8) + ECE 166
  • 771 (13/8) Control


Transforming ECE 188, 189, 190 (3rd try)

- 2 tubes of chemically competent top 10

- Spin down, remove liquid

- Resuspend cells in 100μL of CaCl2 50mM

- 4 tubes with 50μL of cells

  • ECE 190 (all)
  • ECE 189 (all)
  • ECE 188 (all)
  • PUC 9 (5μL) [ Control ]

- Ice for 30 mins

- 42°C for 2 mins

- Ice for 2 mins

- Incubate at 37°C for 2 hours

- Plate out on Amp 100 (Neat)

Biobrick E0040 and I13522 (PA)

  • PCR E0040 colonies 4 and 5 after miniprep of plasmid to ensure plasmid identity again
  • Protocol:
    • SDW 7.5μL
    • MasterMix 10μL
    • VR and VF2 1x2μL
    • DNA sample 0.5μL
  • Run on 1.2% SyBr E-gel
  • Lane 2 - 100bp ladder
  • Lane 3 - E0040 colony 4
  • Lane 4 - E0040 colony 5
  • Result: Bands at around 1000bp on both lanes 3 and 4 with a brighter band for lane 3. Correct band as E0040 VF-VR is 958bp
  • Minprep of plasmid E0040 from colony 4 is kept
  • Transform chemically compotent TOP10 with I13522 biobrick extract again
  • Transformation following standard protocols
  • TOP10 with I13522 plated neat and 1/10 on Amp. 100 plates and incubate at 37°C overnight
  • Will pick single colonies tomorrow for PCR analysis and into LB with Amp100
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