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:
 * 2) 2µl Master Mix
 * 3) 3µl 790bp - pBR322orig
 * 4) 3µl 700bp - PCR digest of my part
 * 5) 2µl 1600bp - PCR digest of my part
 * 6) Tube 2:
 * 7) 2µl Master Mix
 * 8) 4µl 790bp - pBR322orig
 * 9) 4µl 700bp - PCR digest of my part
 * 10) Tube 3:
 * 11) 2µl Master Mix
 * 12) 4µl 790bp - pBR322orig
 * 13) 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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