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From 2010.igem.org

Himika

Today I did a plasmid switch for E0040 (GFP). I plasmid switched E0040 from pSB1A2 to pSB1AC3. I did this plasmid switch because B0034 and E0040 are both in A plasmids. I wish to construct a circuit with B0034-E0040 which would not give me a selection pressure. After transformation of E0040 the future construction will give me selection pressure in order to select for the construct.

Procedure:

• Both E0040 and pSB1AC3 was cut with EcoRI and PstI in invitrogen buffer II
• Insert E0040
  • 3.5 uL buffer II
  • 0.5 uL EcoRI and 0.5uL PstI
  • 6.0 uL DNA with [102.2 ng/uL]
  • 24.5 uL water

• Vector
  • 3.5 uL buffer II
  • 0.5 uL of EcoRI and 0.5uL PstI
  • 2 uL DNA with [180.0 ng/uL]
  • 28.5 uL water

The restriction digest was allowed to sit in the 37 degree hot water bath for 1 hour. Then 5uL of the vector and insert were put in a new tube and 10 uL of quick ligase buffer was added along with 1uL of buffer. This solution was allowed to sit in room temperature for 10 minutes and then they were transformed in top 10 competent cells and plated on Chloromphenicol plates.

I also made overnight cultures for I03504 and I03507 in Amp broth.