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 :

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