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Week of 6/7
From 2010.igem.org
Contents |
June 9th
- Flasks mini prepped
-K091107 (pLux/cI Hybrid Promoter) -R0061 (Promoter (HSL-mediated luxR repressor) -R0063 (Promoter (luxR & HSL regulated -- lux pL) -K091146 (pLas/Lux Hybrid Promoter) -R1062 (Promoter, Standard (luxR and HSL regulated -- lux pR) -K145150 (Hybrid promoter: HSL-LuxR activated) -C0261 (luxI + RBS) -J37033 (RBS + LuxR) -J23108 (Constitutive promoter) -J23116 (Constitutive promoter) -C0062 (luxR repressor/activator)
- All of the mini preps were digested with the following:
-K091107 (ES) -R0061 (ES) -R0063 (ES) -K094116 (ES) -R1062 (ES) -K145150 (ES) -C0261 (XP) -J37033 (XP) -J23116 (ES) -C0062 (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.
- Following the digest we ran a Ladder, C0261, RFP + RBS, and J23116 on a ten-well gel. In Well # 1 is the ladder, Well # 3,# 4 have C0261, # 6,# 7 have RFP + RBS, and # 9,# 10 have J23116.
- As you can see, C0261 (LuxI) did not show up in the gel. It was supposed to have been phosphotased; however, we did not do so.
- We extracted the visible bands, purified, ligated, and transformed.
June 10th
- Flasks mini prepped
-K274100 (CrtEBI with rbs) -C0261 (luxI + RBS) -J23108 (Constitutive promoter) -J23116 (Constitutive promoter)
- All of the mini preps were digested with the following:
-K274100 (XP) -C0261 (XP) -J23108 (ES) (phosphotase) -J23116 (ES) (phosphotase)
- The digests were placed into the 37 degree Celsius water bath for 1 hour and then K274100 and C0261 were heat killed for 20 minutes in an 80 degree Celsius water bath. This was done to stop the reaction. J23108 and J23116 (Constitutive promoters) were phosphotased and placed back into the 37 degree celsius water bath for another hour before being heat killed for 20 minutes in the 80 degree celsius water bath.
- After the 80 minutes of digestion reaction, the DNA was run on a gel. We ran a ladder, C0261, K274100, J37033, and another C0261 because the Constitutive Promoters were being Phosphotased.
- As you can clearly (or not so clearly) see, the gel did not turn out as planned. Initially we believed the gel was old and that was the issue; however, we made a new batch of SYBR Green and future gels were much better.
Later That Day
- We used a new batch of SYBR green and we reran everything from the earlier gel.
- It is much better except that the C0261 and J37033 still did not show up. The ladder on the right has the new SYBR green and the ladder on the left used the old SYBR green.
June 11th
- On this day we made Electrocompetent cells for the first time by ourselves. Being inexperienced with this procedure it took longer than planned. Those of us not making the cells, made media and plates.
