IGEM:Cambridge/2008/Notebook/Bacillus/2008/07/29

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Result from yesterday

  • Result from the gel (29/07/2008)
  • Lane8 : ppL82 (plate)
  • Lane9 : ppL82 (tube)
  • Lane 10 : I746001
  • Lane 11 : I746101
  • Lane 12 : hyperladderI
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The ladder seems to be wrong. So it was really difficult to ckeck the size of our plasmid ppL82. To make, that, we assume that the size of our biobricks was ok, and we estimate the size of our plasmid. The plasmid ppL82 seems to have the right size, but we will have to check again to be able to use a ladder.

  • Competence of B.S. kept on the bench

We observed B.S. with a microscope. Problem, all B.S. seems to be dead!

Possible reasons of this problem :

- Problem with the vector (it could be a wrong vector, we are not really sure)

- Quantity of DNA, we added 0.5μg. The protocol was 1μg, but normally it should be ok.

- B.S. may not be competent (we have to test motility with microscope)

- Cells could be dead : replate them

- Try to add tryptophan in the medium


Probable reason : we forgot to dilute the medium B, that's why it did not work


  • Results from transformation plates (B.S.)


There are 2 colonies on a plate, but it does not look like Bacillus. Moreover, there is also a colony on our negative control plate. SO it must be contamination (resistant to CM!). We will do the transformation again.


Wet Work

  • Prepare medium for transformation of B.S.


- Preparation of medium B: 10mL of medium A and 0.2mL of 50mMCaCl22(H2O) + 250mMgCl26(H2O)

- Preparation of medium A with tryptophan : 81mL of SDW, 9mL of 10X Bacillus salts, 10mL of 10X Medium A base and 0.1mL of Tryptophan (11mg/mL)


  • Check plasmid PNZ8901


- Plasmid miniprep (same protocol with 60μL of elution buffer)

- Digest : with PstI and SalI from Biolabs, and Buffer 3 (add 15μL of DNA)

- Gel (17μL of sample and 3μL of dye)


Results from the gel

  • Lane9 : PNZ8901
  • Lane10 : HyperladderI


Photoh.gif

The sizes we expected were about 1100kb and 2100kb. The sizes should be ok!