IGEM:Cambridge/2008/Notebook/Bacillus/2008/07/30

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Wet Work

  • Transforming Bacillus Subtilis with medium A and medium A + tryptophan

Medium A

Time (min) 0 20 40 60 73 85 95 105
OD650 0.1394 0.1370 0.1660 0.2304 0.2778 0.3138 0.3373 0.3583

For this medium, the curb stop to increase logarithmically at 100min.


Medium A + tryptophan

Time (min) 0 20 40 60 73 85 95
OD650 0.1493 0.1636 0.2080 0.2909 0.3512 0.4002 0.4165

For this medium, the curb stop to increase logarithmically at 90min.


- Incubate 90min at 37°C by shaking

- Prepare 7 tubes for medium A and 7 tubes for medium A + tryptophan : add 0.45mL of prewarmed medium B and 0.05mL of culture and incubate 90min at 37°C by shaking.

- Transform B.S. by adding DNA and incubate 30min at 37°C

We have one transformation with ppL82 (add 7.5μL of DNA), one with PNZ8901 (add 10μL of DNA) and one negative control without DNA.

- Plate 500μL on Cm plate (35μg/mL)


- For the remaining tubes, in one, add 60μL of glycerol and keep it in the freezer and keep the other one on the bench (for each medium)

  • Checking our big stock of biobricks and PNZ plasmid


We did big culture of biobricks and PNZ8901 plasmid to be able to make stocks. We want to check them before making stocks.

- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks

- Measure DNA concentration in our samples to decide the volume of DNA we have to add in our preparation

260/280 DNA concentration (ng/nl)
I746001 1.79 50.5
I746101 1.87 54.3
PNZ8901 1.84 87


- Digest

For I746001 and I746101 For PNZ8901 plasmid
14μL of SDW 14μL of SDW
2μL of 10X Fast Digest Buffer 2μL of Buffer 3 (Biolabs)
2μL of DNA of biobricks 2μL of DNA of PNZ8901
1μL of EcoRI 1μL of PstI
1μL of SpeI 1μL of SalI


- Gel

  • Results from this gel
  • Lane 9 : I746001
  • Lane 10 : I746101
  • Lane 11 : PNZ8901
  • Lane 12 : HyperladderI
Photob.gif

Everthing is really too big! There is a problem, either dimerization, either contamination, either a problem in our work. So we are going to run a new gel.

  • New gel to check

- Miniprep plasmid from growth bottles (I746001, I746101 and PNZ8901); from plates (I746001, I746101 and PNZ8901).

- Do single digest for PNZ8901 (one with PstI, one with SalI)

- Run a gel with : PNZ8901 from friday, PNZ8901 digest with PstI, PNZ8901 digest with SalI, 3 samples from growth bottles, 3 samples from plates (to check the size of the uncut vectors)

  • Result from this second gel
Photoc.gif

The ladders are really bad! But the size of our biobricks and plasmid are too big! So we can not trust these big cultures. They is a problem. With tests from the first week, we know that biobricks are right, so we are going to grow new cultures from these first cultures, to make stocks.

Concerning the vectors, they are from last year, so we are not sure of what they are. Since we ordered new well defined vectors (we should receive them on friday), we will use them in the next steps to be sure of our work.