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Result from yesterday
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.
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
- 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)
- 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
The sizes we expected were about 1100kb and 2100kb. The sizes should be ok!