IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/03

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Checking the insert of promoters

We ligated promoters into death vector, and transforned into Top 10. Our transformation worked, but it was hard to say with the result of yesterday if we had the good insert. Moreover, it is possible that we inverted Pupp and Pspac. So we are going to make a new single colony PCR with the primers of promoters (we used the same single colonies than yesterday).

We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.

Picture of the gel with the different promoters


promoter colony primers name
Pspac 1 Pspac primers A
Pspac 1 Pupp primers B
Pspac 2 Pspac primers C
Pspac 2 Pupp primers D
Pupp 1 Pupp primers E
Pupp 1 Pspac primers F
Pupp 3 Pupp primers G
Pupp 3 Pspac primers H

- Single colony PCR : add 10μL of SDW, 5μL of MM, 1μL + 1μL of primers, 3μL of cells

- Gel

  • lane 3 : hyperladderI
  • lane 4 : Pspac (colony1) with Pspac primers
  • lane 5 : Pspac (colony1) with Pupp primers
  • lane 6 : Pspac (colony2) with Pspac primers
  • lane 7 : Pspac (colony2) with Pupp primers
  • lane 8 : Pupp (colony1) with Pupp primers
  • lane 9 : Pupp (colony1) with Pspac primers
  • lane 10 : Pupp (colony3) with Pupp primers
  • lane 11 : Pupp (colony3) with Pspac primers
  • lane 12 : HyperladderI

- Results

promoter colony primers observation Conclusion
Pspac 1 Pspac primers nothing it is in fact Pupp
Pspac 1 Pupp primers band (about 280bp) Pupp OK
Pspac 2 Pspac primers nothing it is in fact Pupp
Pspac 2 Pupp primers band (about 280bp) Pupp OK
Pupp 1 Pupp primers nothing it is in fact Pspac
Pupp 1 Pspac primers band (about 180bp) Pspac OK
Pupp 3 Pupp primers nothing it is in fact Pspac
Pupp 3 Pspac primers band (about 180bp) Pspac OK

Make some stock of our new biobricks

  • Pupp (colony1) into the death vector, transformed into TOP10
  • Pspac (colony1) into the death vector, transformed into TOP10
  • agrD (colony5) into the death vector, transformed into TOP10

- Grow this single colony into 10mL of LB (Cm35)


Transformation of new "biobricks"

- transform into E.coli :

  • agrA
  • agrB
  • agrC
  • Pxyl


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