Week 5
From 2007.igem.org
- 07/30/07
- We check bacterial growth curve (link).
- Transformation for C0012, J52034 and B0015.
- We get along with the ligation R0051+I13507 (I763007) (vector 2 ul, insert 6 ul) and the ligation J04500+ C0051 (I763005)(vector 2 ul, insert 10 ul).
- 07/31/07
- There are no colonies for C0012 , so we inoculate I52034 and B0015 colony in 5 ml.
- We strake on plates 30/07 ligations.
- We get along with the J04431 fluorescence test:
-From the glycerol stock we sprout 1 ml at 37°C for 1 hour in agitation.
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210 min and OD=0.5.
- We decide to repeat the test to understand if we can switch off the fluorescent cells with a higher amount of glucose.
We will perform this test on 07/08.
- 08/01/07
-Miniprep of I52034 and B0015;
-Digestion of B0015 with Xba/Pst1;
-Digestion of I52034 with Spe/Pst1;
- Gel extraction (we can’t see B0015);
-R0015+I13507(I763007) ligation which doesn’t give colonies so we transform with the other 10 ul of ligation;
-J04500+C0051(I763005)ligation which gives colonies so we inoculate in 5ml;
-Transformation for C0012 (4ul) and for J22101 (2ul);
-A new R0051+ I13507 (I763007) (vector 4ul, insert 8ul) ligation.
- 08/02/07
-Miniprep of J04500+C0051 (I763005);
-Control digestion of 5 ul with Eco/Pst1;
-A new B0015 digestion with Xba/Pst1;
-Run on electroforesis gel. We have problems with gel because we don’t see the band;
-Band extraction for B0015 e I763005;
-There are no colonies for C0012, but there are colonies for R0051 + I13507(I763007). So, we identify the correct ligation protocol;
- R0010, P0412, C0012 transformation;
-Inoculation in 5 ml for R0051 + I13507 and for J22101;
- R0051 + I13507 (I763007) fluorescence test to check if the RFP filter works properly.
- 08/03/07
- We get along with:
-Miniprep for J22101 and for R0051+ I13507 (I763007);
- J22101 digestion with Xba/Pst1 ;
- Control digestion of R0051 + I13507 (I763007);
- J04500+C0051 (I763005) and B0015and J22101 and R0051 + I13507 (I763007) run on electroforesis gel and J04500+C0051 (I763005) and J22101 extraction;
-Glycerol stocks preparation;