IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/06

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Result from transformation

PLATES HAD BEEN BINNED!! OOPS

Check Vectors

- Plasmid miniprep for ECE176 (from 05/08 LB stock and from 04/08 LB stock with a second inoculation)

We want to check uncut plasmid, single and double digest for ECE147, ECE149, ECE150, ECE153, ECE162, ECE172, ECE176. In order to have enough DNA (to see the bands), we will add 1μg of DNA to make single digest, and wr will incubate during 1h (instead of only 10min).

  • Nanodrop
Vetor 260/280 μg/mL
ECE147 1.67 50.8
ECE149 1.66 51.9
ECE150 1.78 107.0
ECE153 1.68 31.2
ECE162 1.56 33.1
ECE172 (04/08) 1.64 22.8
ECE172 (05/08) 1.89 213.1
ECE176 (04/08) 1.89 111.1
ECE176 (05/08) 1.83 96.2

We made singe digest for the samples from 05/08 only.

- Double digest (1 hour of incubation in water bath at 37°C)

- Gel

Gel1

  • Lane1 : HyperladderI
  • Lane2 : ECE147 with EcoRI
  • Lane3 : ECE147 with HindIII
  • Lane4 : ECE149 with SpeI
  • Lane5 : ECE149 with PstI
  • Lane6 : ECE150 with SpeI
  • Lane7 : ECE150 with HindIII
  • Lane8 : ECE153 with SalI
  • Lane9 : ECE162 with SalI
  • Lane10 : ECE172 with HindIII
  • Lane11 : ECE176 with EcoRI
  • Lane12 : HyperladderI

Gel2

  • Lane1 : HyperladderI
  • Lane2 : ECE176 with XbaI
  • Lane3 : ECE176 double digest
  • Lane4 : ECE150 double digest
  • Lane5 : ECE149 double digest
  • Lane6 : ECE147 double digest
  • Lane7 : HyperladderI
  • Lane8 : ECE149 uncut
  • Lane9 : ECE153 uncut
  • Lane10 : ECE172 uncut
  • Lane11 : supercoiled ladder
  • Results

Our supercoiled ladder was very bad, so it was impossible to conclude for uncut vectors.

- ECE147, ECE 150, ECE153, ECE 176 single digest : ok

- ECE149 : problem, 2 cutting sites for PstI (only one in the sequence)

- ECE162 : only one band whereas SalI should have 2 cutting sites

- ECE172 : wrong sizes!!!

- Double digest for ECE176 : ok!

- Double digest for ECE 150, 149 and 147 : only one band!!!

There may be a problem when we do the double digest by adding two single digest. We will try again with a direct double digest.


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