IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/09/22

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dspB Track

Gel verification on digestion

  • Protocol: gel verification protocol in Protocol (SOP)
  • Machine conditions: 0.5x TBE buffer, 110V, 60min

Gel orientation:

Gel orientation
DG100bp laddersuf1 kb ladder

Results:
File:100921 dspB 36.tif

  • DspB isn't cutting. Second try.
  • Next step (troubleshooting): try using the RD buffer that has 100% activity for each enzyme in a reaction (NEbuffer2 = 100% for all of them already, but try using NEbuffer 4)

Re-Restriction Digest

  • Protocol: "digest" protocol from biobrick
Restriction Digest
REAGENTS1 RXN VOLUME (uL)
Buffer 42
BSA0.2
DNAx
ddH207.8
Total20
  • x = amount of DNA
  • DNA:
    • Cut terminator (suffix) at E/X (use x = 15uL)
    • Cut D and G at E/S (use x = 10uL)
  • Add 1uL of each appropriate enzyme

Tubes: C4, D4, G4, H4, C2, H2, term4

  • 4 = used buffer4; 2 = used buffer2

Gel verification on digestion

  • Protocol: gel verification protocol in Protocol (SOP)
  • Machine conditions: 0.5x TBE buffer, 110V, 60min

Gel orientation:

Gel orientation
C2C4D4100bp ladderH2H4G4term41 kb ladder

Results:
File:100922 dspB 37.tif

  • Still single cuts
    • Tried buffer 2 and 4 -> both didn't work
    • SpeI dead?

Re-Restriction Digest with new SpeI

  • Protocol: "digest" protocol from biobrick
Restriction Digest
REAGENTS1 RXN VOLUME (uL)
Buffer 42
BSA0.2
DNA8
ddH207.8
Total20
  • DNA:
    • Cut D and G at E/S
  • Add 1uL of each appropriate enzyme

Gel verification on digestion

  • Protocol: gel verification protocol in Protocol (SOP)
  • Machine conditions: 0.5x TBE buffer, 100V, 60min

Gel orientation:

Gel orientation
CD100bp ladderHG1 kb ladder

Results:
[[Image:‎|300px]]

  • No cuts......
  • PROBLEM: construct is E-X-dspB-P-S-P