Team:UCLA/Notebook/Spider Silk Genetics/24 August 2015

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8/24/2015

• Gel extraction for M1/2[1:1]-12, M1/2[2:1]-12

Digestion for M1/2[1:1]-12, M1/2[2:1]-12

• 2 reactions for each.
• Digest one reaction with E, P for cloning into pSB1C3.
• Digest one reaction with X, P for cloning into BBa_K525998.
• Digest at 37 C for 1.5 hrs, heat kill at 65 C for 20 min.
• Products were PCR purified after digestion.

ICA for M1/2[1:2]-12

• Used our finalized protocol.

Ligation

• Ligated M1-12; M1/2[1:1]-12, M12[2:1]-12 in 20 uL reactions
  • M1-12 was ligated only into pSB1C3.
  • The others were ligated into pSB1C3 and BBa_K525998.

Transformation

• Ligation products for M1-12(1C3), M1/2[1:1]-12(1C3), and M1/2[2:1]-12(1C3) were transformed into electrocompetent DH5(alpha) cells.
  • DNA was dialyzed against water prior to transformation
• Ligation products for M1/2[1:1]-12(T7) and M1/2[2:1]-12(T7) were transformed into chemically competent BL21(DE3) cells.