Gel electrophoresis for overnight digested products. Please refer to 10 June 2008 notebook (Reminder: the ratio of loading dye to test sample is 1:5)
Result: + RBS 32 and 34 that were digested with XbaI and SpeI showed correct bands at 2kb. Control experiments (RBS 32 and 34 digested with EcoRI and PstI) also showed correct bands at 2kb. + E7-1 PCR insert that was digested with XbaI and SpeI did not show any band, thus unsuccessful.
Gel electrophoresis for confirmation after Gel extraction (together with gel electrophoresis of T7 promoter). Result:All the products showed correct 2kb bands. Also, as expected, T7 promoter didn't show any additional band as the pT7 gene is too small and probably run out of the gel.
Ligation of E7-0 insert into RBS 32 empty plasmid vector.
Insert 1
E7-0 PCR insert
16ul
Vector 2
empty plasmid vector(from RBS 32)
2ul
ligase buffer
T4 ligase buffer
2ul
ligase enzyme
T4 ligase
1ul
Total
21ul
(After adding above amounts, incubate the mixtures at 4oC for at least 4-5 hours)
Transform ligated E7 (new biobrick part) into Top10 competent cells.
Transform LacI-GFP into BL-91 as control.
Chin Chong
Realised that the fluroscence meter requires a Black based 96-well for fluroscence measurements
clear based wells will have noise and interference from adjacent wells
Well should also be F-bottom for better results
Ordered a new batch of Black Uclear from Greiner Product no. 655 090
Stopped characterization tests till the new plates have arrive
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