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From 2010.igem.org

Contents 1 June 14th 2 June 15th 3 June 16th 4 June 17th

June 14th

• Flasks mini prepped

   -J37033 (LuxR + RBS)
   -C0261 (LuxI + RBS)
   -R0061 (Lux Promoter)
   -J23116 (Constitutive Promoter)
   -R0063 (Lux Promoter)
   -K145150 (Lux Promoter)
   -K091146 (Lux Promoter)
   -K274100 (Pigment)
   -J23108 (Constitutive Promoter)

• All of the mini preps were digested with the following:

   -J37033 (XP)
   -C0261 (XP)
   -R0061 (ES)
   -J23116 (ES)
   -R0063 (ES)
   -K145150 (ES)
   -K091146 (ES)
   -K274100 (ES)
   -J23108 (ES)

• We ran the digest as usual: 1 hour in the water bath, 20 minutes heat kill. We did not phosphotase any of these as you can see all of these parts appear on the gel.

• The order was Ladder, J23108, K274100, K091146, K145150, R0063, J23116, R0061, C0261, and J37033.

• This gel came out well with the exception of K145150.

• We then cut out each available band, purified, ligated, and finally transformed.

June 15th

• Flasks mini prepped

   -J37033 (LuxR + RBS)
   -C0261 (LuxI + RBS)

• All of the mini preps were digested with the following enzymes:

   -J37033 (XP)
   -C0261 (XP)

• The digests were put into a 37 degree Celsius water bath for an hour. The reaction was heat killed for 20 minutes in an 80 degree Celsius water bath.

• The two parts were then run on a gel in this order: Ladder, PFTV (another student's project), C0261, and J37033.

• This time the parts came out very vibrantly. We extracted C0261, did the gel purification, ligated, and transformed.

June 16th

• Flasks mini prepped

   -R0010 (pLac)
   -C0062 (LuxR)

• All of the mini preps were digested with the following enzymes:

   -R0010 (XP)
   -C0062 (XP)

• The digests were put into a 37 degree Celsius water bath for an hour. The reaction was then heat killed for 20 minutes in an 80 degree Celsius water bath.

• After the heat kill, an Agarose gel was run. The parts run on the gel were Ladder, R0010, and C0062.

• After the gel was finished running, we extracted the necessary bands and purified them.

• We made notes of possible ligations:

   -Lux Promoters <-> C0261 (LuxI)
   -Lux Promoters <-> K274100 (Pigment)
   -Constitutive Promoters <-> J37033 (Lux R)
   -CI Promoters (K091107) <-> Lysis (K112022, K112808)

• Goal for June 17th:

   -Ligate B0034 <-> C0051
                     C0012
                     C0062

June 17th

• Flasks mini prepped:

   -J24679
   -K091107
   -R0061
   -J37033
   -K081007
   -LacI + RBS

• All of the mini preps were digested with the following enzymes:

   -K081007 (XP) (Phosphatase)
   -K091107 (SP) 
   -J37033 (XP) (Phosphatase)
   -R0061 (SP)
   -J37033 (PCR) (XP)
   -C0261 (PCR) (XP)

• The digests were put into a 37 degree Celsius water bath for one hour. The reaction was then heat killed for 20 minutes in an 80 degree Celsius water bath.

• After the heat kill, an agarose gel was run in the order of Ladder, J37033 (PCR), K274100, C0261 (PCR), R0061, K091107, and then P, T, V (another project).

• Once the gel was finished we extracted and purified the parts.

• We made competent cells, SOB media, and Tet plates.

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