User:Howard Boland/Notebook/Art from Synthetic Biology/2010/11/23

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Check plate overnight growth with H2O2 disk check

The plate showed growth only around 1/1000 and 1/10000 dilution of 33% H2O2 concentration but I was not observing GFP under UV-light. The following concentrations can be observed by reading the disk starting with the one below the arrow and reading clockwise.

  1. Disk 1: 1/1
  2. Disk 2: 1/10
  3. Disk 3: 1/100
  4. Disk 4: 1/1000
  5. Disk 5: 1/10000
  6. Centre Disk: Water / Control

Digestion pUA66 and katE with PstI

2xMaster Mix

  1. 1µl 100xBSA
  2. 10µl 10xNEB#3
  3. 4µl PstI
  4. 25µl H2O

For each of two reaction tubes add 20µl Master Mix

  1. Tube 1: 30µl pUA66
  2. Tube 2: 20µl katE

Incubate in 37ºC for 2 hours

Digestion pSense66 XhoI/BamHI

  1. 0.5µl 100xBSA
  2. 5µl 10xNEB#3
  3. 1µl XhoI
  4. 1µl BamHI
  5. 12.5µl H2O
  6. 30µl pSense66

Incubate in 37ºC for 2 hours

Gel of Digestion

  1. Lane 1: 10µl 1kb Quick Ladder
  2. Lane 2: 10µl pUA66katE - PstI
  3. Lane 3: 50µl pUA66 - PstI
  4. Lane 4: BLANK
  5. Lane 5: 50µl katE - PstI
  6. Lane 6: BLANK
  7. Lane 7: 50µl pSense66 - XhoI / BamHI
  8. Lane 8: Blank

Gel Picture

Digestion pSense66 XhoI/BamHI

  1. 0.5µl 100xBSA
  2. 5µl 10xNEB#3
  3. 1µl XhoI
  4. 1µl BamHI
  5. 12.5µl H2O
  6. 30µl pSense66

Incubate in 37ºC overnight, for 16 hours


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