IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/07/28: Difference between revisions

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[image:Bacillus_competence_log_graph.png|center|Log of OD650 versus time for Competent Bacillus Cell Culture]


At, t0 = 70min, log growth seems to stop.
At, t0 = 70min, log growth seems to stop.

Revision as of 04:38, 30 July 2008

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Results from previous days

  • Result of the gel

We wanted to check the size of our biobricks. For I746101, we have a band of about 3000kb, which is the size of the vectorm and a band of about 2000kb. The size of the biobrick is 2057kp, so it should be ok.

For I746001, we hqve the same band of 3000kb (the vector) and a band of about 1000kb. The size of the biobrick is 896kb.

So, the size of our biobricks are ok.

  • Results of antibiotic plates from yesterday

For Cm, 35μg/mL should be enough to kill B.S.

For Amp,

Wet Work

  • Check plasmid ppL82

We had 2 samples of ppL82 in DH5α in LB solution (~4mL), one from the frozen glycerol tube and one from a colony picked on a plate. Normally plasmid and cells should not be kept in freezer. So, we want to extract this plasmid, check its size and then keep it in the fridge. We do that for the 2 different samples.


- Plasmid Miniprep (standard protocol)

- Test the concentration of DNA in each tube

  1. ppL82 plate : 89.8ng/mL
  2. ppL82 tube : 71ng/mL

- Digest

  1. For biobricks I746001 and I746101, use digest enzymes EcoRI and SpeI, and 2μL of DNA
    Add 15μL of SDW, 2μL of 10X Fast Digest Buffer, 2μL of DNA, 1μL of EcoRI and 1μL of SpeI
  2. For ppL82 (from plate and tube), use digest enzymes BamHI and PstI and add 15μL of DNA
    Add 2μL of SDW, 2μL of 10X Fast Digest Buffer, 15μL of DNA, 1μL of BamHI and 1μL of PstI
  3. Microfuge tubes
  4. Incubate 10 min at 37°C
  5. Heat shock for 5min at 80°C

- Gel : add 4μL of dye and 21μL of samples



  • Preparation of different stocks of strains and plasmids


PNZ8901

- Plate a single colony (from 23/07/08) on a Cm plate

- Incubate at 37°C


- Grow the entire frozen glycerol tube in 20mL of LB without antibiotic to check if this stock is still good (we will check the plasmid on a gel tomorrow)

- Incubate at 37°C


MC1061

-Pick e single colony of MC1061 (from 22/07/08) and put it in 10mL of LB (to make glycerol stocks)

-Incubate at 37°C


1A1

- Add 100μL of LB+1A (mix of friday) and 10mL of LB

- Incubate at 37°C


  • Make B.S. 1A1 competent and transform them

- Prepare Medium A

Add 81mL of SDW, 10mL of 10X Medium A base and 9mL of 10X Bacillus salts


- Make B.S. 1A1 competent

  1. In 10mL of medium A and add about 10 colonies of B.S.
  2. Check the OD650, you should have an OD between 0.1 and 0.2. t0 for OD = 0.1876
  3. Check the OD650 every 20min and plot OD against time on semi-log paper. When the point at the culture leaves log growth, it is ok
Time (min) 0 20 40 60 70 80
OD650 0.1876 0.2074 0.3282 0.4545 0.4895 0.5040

[image:Bacillus_competence_log_graph.png|center|Log of OD650 versus time for Competent Bacillus Cell Culture]

At, t0 = 70min, log growth seems to stop.

  1. Incubate at 37°C 90min after t0
  2. Warn 10 Ependorf tubes with 0.45mL of Medium B
  3. Add 50μL of culture in each tube of Medium B at t90
  4. Incubate the diluted culture at 37°C with vigorous aeration for 90min

- Storage : we want to store 7 tubes

6 tubes in the freezer (with 60μL of glycerol)

1 tube on the bench

- Transformation

  1. We make ppL82 from plate, ppL82 from tube, and a control
  2. Add 0.5μg of DNA (15μL)
  3. Incubate at 37°C for 30min
  4. Plate on Cm plates


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