Team:UNIPV-Pavia/Notebook/Week12

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Notebook

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Week 15	Week 16	Week 17	Week 18	Week 19	Week 20	Week 21
Week 22	Week 23	Week 24

08/4/08

J23100-B0030-C0040-B1006 (E-S)

R0040 (E-X)

Ligation: J23100-B0030-C0040-B1006-R0040. We incubated ligation at 16°C overnight.

Last week J23100-B0030-C0012-B1006-R0010 colony PCR gave a bad result. For this reason, we decided to perform colony PCR only for:
- B0030-C0061-B1006-R0062-B0030-E0040-B1006 (7 colonies)
- J23100-B0030-C0012-B1006-R0010 (6 colonies)

Marker 1Kb, blank, J23100-B0030-C0012-B1006-R0010 (6 colonies), B0030-C0061-B1006-R0062-B0030-E0040-B1006 (7 colonies)

Gel result:
- B0030-C0061-B1006-R0062-B0030-E0040-B1006 (1st colony, but it was not pure. We decided to prepare single colonies plate for it)
- J23100-B0030-C0012-B1006-R0010 (2nd colony)

08/4/08

We transformed J23100-B0030-C0040-B1006-R0040 overnight ligation. We plated transformed bacteria and incubated plate at 37°C overnight.

We infected 9 ml of LB + Amp with 30 µl of C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-R0062-B0030-E0040-B1006 (1st col) (see "Parts" section for our nomenclature).

Single colonies plates for:
- B0030-C0061-B1006-R0062-B0030-E0040-B1006 ("Lig.b")
- B0030-C0078-B1006-R0079-B0030-E0040-B1006 ("Lig.c")
- B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006 ("Lig.d")

08/5/08

Glycerol stocks/miniprep for C0062, Lig.12, Lig.22, Lig.30, Lig.27(2nd col), B0030-C0061-B1006-R0062-B0030-E0040-B1006(1).

We sent C0062, Lig.12, Lig.22 and Lig.30 purified plasmids to Primm for sequencing: all these parts contain BBa_C0062.

Colony PCR for a (7 colonies), Lig.b(single colonies)(6 colonies), Lig.c(single colonies)(6 colonies), Lig.d(single colonies)(6 colonies).

Marker 1Kb, blank, Lig.a (7 colonies), Lig.b (6 colonies), Marker 1Kb, Lig.c (6 colonies), Lig.d (6 colonies)

Gel results were not so clear: the length of some fragments was not expected and there were some contaminants. Maybe those parts were too long for our PCR reaction. We decided to grow 9 ml cultures for some of those colonies, to extract plasmids, to cut them and to check their length in a new run. We chose:
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062-B0030-E1010-B1006 (1, 4, 6, 7)
- B0030-C0061-B1006-R0062-B0030-E0040-B1006 (no colony was chosen: we already had them and this run didn't show any 100% pure colony)
- B0030-C0078-B1006-R0079-B0030-E0040-B1006 (5)
- B0030-C0061-B0030-C0079-B1006-R0079-B0030-E0040-B1006 (2)

08/6/08

Single colonies plate for J23100-B0030-C0040-B1006-R0040, because where were too many bacteria on its plate.

We cut these 6 plasmids and Lig.b in (E-P) (5 µl of DNA in a final reaction volume of 20 µl).

Marker 1Kb, Lig.a (1), Lig.a (4), Lig.a (6), Lig.a (7), Lig.b (1), Lig.c (5), Lig.d (2)

08/7/08

Qualitative fluorescence tests for Lig.b and Lig.c. Results are shown in The Project section (Experiments).

Colony PCR for J23100-B0030-C0040-B1006-R0040 single colonies plate (screening on 6 colonies).

Marker 1Kb, blank, J23100-B0030-C0040-B1006-R0040 (6 colonies)