IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/15: Difference between revisions

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- Make master mix enough for 4RXNs
- Make master mix enough for 4RXNs


* '''Digest and Ligate Agr A and C to pSB4C5'''
Previous PCR products of Agr A and C were digested using Fermentas Fast Digest - EcoRI and PstI according to their protocol except a longer time was given to digest.
3uL of AgrA was used and 4.4 uL of AgrC was used and incubated for 40minutes.
10uL totaling to 1ug of pSB4C5 plasmid was also digested and incubated for 10 minutes.
The digestion was visualized on a 0.8% Agarose gel and produced expected sizes:
Plasmid: approx 3bk
Agr A: approx 700bp
Agr C: approx 1300bp




Revision as of 10:19, 19 August 2008

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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.

Lab work

  • 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)


  • PCRing out promoters

- 4 different promoters : Pxyl, Pspc, Ppac, Pupp

- Make master mix enough for 4RXNs

  • Digest and Ligate Agr A and C to pSB4C5

Previous PCR products of Agr A and C were digested using Fermentas Fast Digest - EcoRI and PstI according to their protocol except a longer time was given to digest.

3uL of AgrA was used and 4.4 uL of AgrC was used and incubated for 40minutes.

10uL totaling to 1ug of pSB4C5 plasmid was also digested and incubated for 10 minutes.

The digestion was visualized on a 0.8% Agarose gel and produced expected sizes: Plasmid: approx 3bk Agr A: approx 700bp Agr C: approx 1300bp




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