From 2008.igem.org
Wednesday 11 June
- Morning:
- Choon Kit, Hung:
- 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 extraction of E7-0 insert (digested with XbaI and SpeI on Tuesday 10 June), RBS 32 and RBS 34 empty plasmid vectors (digested with XbaI and SpeI on Tuesday 11 June) using QIAgen Gel Extraction Kit.
- 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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