Team:UCLA/Notebook/Spider Silk Genetics/9 July 2015

iGEM UCLA




7/9/2015

Ligation for 3-, 6- mer, and MaSp2 Seq AB

  • Used the pieces that were digested yesterday.
  • Used [http://www.insilico.uni-duesseldorf.de/Lig_Input.html ligation calculator] to determine proper amounts of insert and vector.
    • Vector size: 2200 bp
    • Vector amount: 50 ng
    • 3:1 insert to vector ratio
    • Insert size:
      • 3-mer: 406 bp -> 27.68 ng
      • 6-mer: 712 bp -> 48.55 ng
      • MaSp2 Seq AB: 170 bp -> 11.59 ng
  • ligate in 20 uL reaction
3-mer 6-mer MaSp2 Seq AB
T4 ligase 1 uL 1 uL 1 uL
Insert 2 uL 7 uL 0.5 uL
Vector 1 uL 1 uL 1 uL
10x T4 Ligase Buffer 2 uL 2 uL 2 uL
ddH2O 14 uL 8 uL 15.5 uL
Total 20 uL 20 uL 20 uL
25 C 25 min
65 C 10 min
12 C hold

Small Scale Liquid Culture for BBa_525998 and BL21(DE3)

  • set up 6 ml cultures for BBa_525998 and BL21(DE3) with chloramphenicol for BBa_525998 and no selection for BL21(DE3).
    • 3 cultures for BBa_525998, plan to miniprep and sequence verify.
    • 1 culture for BL21(DE3), plan to make chemically competent cells.

Drop Dialysis

  • Performed drop dialysis of 2 uL of ligation reactions against ultra-pure water for 10 minutes to remove salts in preparation for electro-transformation.
  • Three drops on same membrane.

Electro-Transformation

  • Electro-transformed the 3-mer, 6-mer, and MaSp2 Seq AB from the drop dialysis. Pulse times listed below.
    • 3-mer: 5.5 ms
    • 6-mer: 5.5 ms
    • MaSp2 Seq AB: 5.5 ms
  • Plated all three onto chloramphenicol plates.