User:Hana Benzeer/Notebook/SGM Summer Project/2011/06/15

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DIGESTION

  1. BBa_E0840
  2. BBa_R0082
  3. BBa_M30109

For the first composite between the E0840 and R0082 parts, as R0082 was chosen as the backbone and E0840 the downstream reporter.

  1. R0082 was digested with P and S
    1. To 30ul of R0082 we added
    2. 0.5ul, 100xBSA
    3. 5ul, buffer 2
    4. 1μL, PstI
    5. 1μL, SpeI
    6. 12.5μL, H2O
    7. The 50μL reaction was placed at 37C for 2 hrs
  2. E0840 was digested with P and X
    1. To 30ul of E0840 we added
    2. 0.5ul, 100xBSA
    3. 5ul, buffer 3
    4. 1μL, PstI
    5. 1μL, XbaI
    6. 12.5μL, H2O
    7. The 50μL reaction was placed at 37C for 2 hrs.

Gel preperation

prepared 1% and 2% agarose gel

1% gel, loading

  1. 1Kb ladder
  2. 50ul, R0082, P/S
  3. 5μL, R0082, P/S
  4. Blank
  5. 5μL, E0840, P/X
  6. Blank
  7. Blank
  8. Blank

2% gel, loading

  1. 100 bp ladder
  2. 50ul, E0840, P/X
  3. 5μL, E0840, P/X
  4. Blank
  5. Blank
  6. Blank
  7. Blank
  8. Blank

Gel purification

the bands were cut with scapel from the gels.

  1. E0840, 900bp
  2. R0082, ~3kb

the method, book

Ligation

To ligate the cognate promnoter R0082 with reporter,E0840.

  1. Add 1μL of R0082 backbone
  2. Add 7μL of E0840 insert
  3. Add 1μL of ligase buffer
  4. Add 1μL of ligase
  5. Incubate at room temp for 2 hrs

Transformation

  1. To 50μL of competent cell (XL-1 Blue)
  2. Add 10μL of ligase reaction
  3. Place for 5 min on ice
  4. Place in the 42°C waterbath for 1 min
  5. Put back on ice for 5 min
  6. Add 250μL Soc
  7. Place in the shaker for 1 hr
  8. Plate on agar with appropriate antibiotic (Ampicillin) using glass beads
  9. Put in the incubater at 37°C




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