Team:UCLA/Notebook/Spider Silk Genetics/15 July 2015

iGEM UCLA




7/15/2015

Sequence Verification

  • ICA MaSp2 3-mer
    • All are good
  • MaSp2 6-mer
    • Sequence failure for 1, 2.
    • Sequence 3 is good.
  • MaSp2 Seq AB
    • 1: BAD: single bp deletion
    • 2: GOOD
    • 3: BAD: 20 bp insert

Restriction Enzyme Digestion, Subcloning Preparation

  • Subcloning 3-mer and 6-mer into BBa_K525998 for expression.
  • Cloning 9-mer into pSB1C3 for sequencing and biobrick making.
Restriction Enzymes Amount to Digest Post-Digest Processing
BBa_K525998 S, P 1 ug of plasmid PCR purify
3-mer X, P 2 ug of plasmid Gel purify
6-mer X, P 2 ug of plasmid Gel purify
9-mer E, P 10 uL of solution PCR purify
  • Digestion was in 50 uL reactions; all used 10x NEBuffer 2.1; used 1 uL of each enzyme.
  • Digest at 37 C for 1.5 hrs, then heat kill at 65 C for 20 min.

Results

Concentration (ng/uL) A 260/280
BBa_K525998 102.46 2.07
MaSp2 3-mer 32.33 2.53
MaSp2 6-mer 31.82 1.70
MaSp2 9-mer 10.28 .1.73

Ligation for Transformation Preparation

  • Ligate MaSp2 3-mer, 6-mer into BBa_K525998.
  • Ligate MaSp2 ICA 9-mer into pSB1C3.
  • Used [http://www.insilico.uni-duesseldorf.de/Lig_Input.html Ligation Calculator] with the following parameters to determine relative DNA amounts:
    • vector size: 2200 bp
    • vector amount: 50 ng
    • insert size:
      • 3-mer: 406 bp
      • 6-mer: 712 bp
      • 9-mer: 1018 bp
    • 3:1 ratio of insert:vector
  • Ligation in 20 uL reactions with 1 uL of T4 Ligase.
    • Ligated at 25 C for 25 min, then heat kill at 65 C for 10 min.

Transformation

  • 2 uL of ligated product MaSp2 3-mer, 6-mer were transformed into BL21(DE3) chemical competent cells.
    • Plated all the bacteria onto chloramphenicol plates.
  • 3 uL of ligated MaSp2 ICA 9-mer were dialyzed against ultra-pure water.
  • Transformed 2 uL of MaSp2 ICA 9-mer into DH5(alpha) electrocompetent cells.
    • Plated all the bacteria onto chloramphenicol plates.