Week 5
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Latest revision as of 15:51, 26 October 2007
- 07/30/07
- We check growth curve for bacteria containing an intermediate plasmid.
- Transformation for C0012, J52034 and B0015.
- Ligation R0051+I13507 (I763007) (vector 2ul, insert 6ul) and ligation J04500+ C0051 (I763005)(vector 2ul, insert 10ul).
- 07/31/07
- There are no colonies for C0012, thus we inoculate I52034 and B0015 colonies in 5ml.
- We strake on plates 30/07 ligations.
- We go along with the J04431 fluorescence test:
-From the glycerol stock we sprout 1ml at 37°C for 1h in agitation.
-Every 15 minutes we check bacterial OD and fluorescence. Cells are fluorescent at t= 210min 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 8th August.
- 08/01/07
-Miniprep of I52034 and B0015;
-B0015 digestion with Xba/Pst1;
-I52034 digestion with Spe/Pst1;
- Gel extraction (we can’t see B0015);
-R0015+I13507(I763007) ligation which doesn’t give colonies thus we transform with the other 10ul 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 electrophoresis 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
-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 electrophoresis gel and J04500+C0051 (I763005) and J22101 extraction;
-Glycerol stocks preparation.