User:Howard Boland/Notebook/Art from Synthetic Biology/2010/11/24

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Gel of Digestion

  1. Lane 1: 10µl 1kb Quick Ladder
  2. Lane 2: 20µl pSense66 - XhoI / BamHI
  3. Lane 3: 5µl pSense66 (Circular)
  4. Lane 4-8: BLANK

Gel Picture

Ligations

I decided to also the my ligation part to see how or which one that works since my 2.3kb product broke down into 1600bp and 700bp.

Master Mix

  1. 3µl Ligase Buffer
  2. 3µl Ligase

I set up three tubes

  1. Tube 1:
    1. 2µl Master Mix
    2. 3µl 790bp - pBR322orig
    3. 3µl 700bp - PCR digest of my part
    4. 2µl 1600bp - PCR digest of my part
  2. Tube 2:
    1. 2µl Master Mix
    2. 4µl 790bp - pBR322orig
    3. 4µl 700bp - PCR digest of my part
  3. Tube 3:
    1. 2µl Master Mix
    2. 4µl 790bp - pBR322orig
    3. 4µl 1600bp - PCR digest of my part

I left the samples in room temperature for 2 hours

Plates

Kanamycin I prepared 7 plates of Agar with Kanamycin using 200ml LB-Agar and 1ml Kanamycin.

Ampicillin / Tetracycline I prepared 7 plates of Agar with AT/AT (Ampicillin/Tetracycline) using 200ml LB-Agar, 200µl Ampicillin and 200µl Tetracycline.

Transformation

I transformed the 3 ligations using heat-shock and also I did a fourth transformation using the part BBa_J45200 taken from F5 of the 2nd plate of the 2010 Spring Distribution Plate.

  1. I placed 50µl competent cells (XL-1 Blue) on ice for 5 minutes
  2. For each of 3 tubes I one ligation reaction and for the last tube I added 1µl BBa_J45200
  3. I waited for 5 minutes
  4. Placed the tube in a 42ºC water bath for 1 minutes
  5. Back on ice for 5 minutes
  6. Added 250µl SOC (warm)
  7. Placed in shaker for 1 hour
  8. Plated the transformation onto 3 Kanamycin plates for the ligation and 1 Ampicillin/Tetracycline plate for the BBa_J45200.

I incubated the plates overnight at 37ºC

Setting-up disk test plate for H2O2

I prepared a new serial dilution of 1/10 and 1/2 H2O of 30% H2O2. (1/1000, 1/10000, 1/20000, 1/40000, 1/80000). A plate was prepared by streaking out glycerol stock over the entire area using a swab. The disk were then placed in a clockwise order from beneath and the plate was left in an incubator to grow overnight.



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