Team:UNIPV-Pavia/Notebook/Week10

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Notebook

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Week 8	Week 9	Week 10	Week 11	Week 12	Week 13	Week 14

Week 10: 07/21/08 - 07/25/08

07/21/08

Colony PCR for BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079: 6 colonies from single colonies plate.

BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079

Gel results: all the 6 picked colonies were true positive! we chose the 5th colont to grow a 9 ml overnight culture.

We also infected 9 ml LB + Amp with 30 µl of BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 and BBa_B0030-BBa_C0061 glycerol stocks to grow two overnight cultures.

Ligation:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (1:2 ratio, 40 ng of vector)
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (30 ng of vector)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006

We incubated ligations at 16°C overnight.

07/22/08

Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.

Glycerol stocks/miniprep for:

BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040	BBa_B0030-BBa_C0061
BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079

We sent BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040 and BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 purified plasmids to Primm for sequencing.

Plasmid digestion for:

BBa_B0030-BBa_C0061 (E-S)

BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 (E-X)

Gel run/cut/gel extraction.

07/23/08

Ligation plates showed colonies!We put ligation plates at +4°C.

Ligation for BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079.

We incubated ligation at 16°C overnight.

07/24/08

Transformation (1 µl) for BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.

Colony PCR for:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006

(We performed one PCR for BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).

Electrophoresis (1.2% agarose) for BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 and BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082:

Marker 1Kb, blank, BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (7 lanes), BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (6 lanes)

Electrophoresis (1% agarose) for BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 and BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006:

Marker 1Kb, BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (7 lanes), BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (7 lanes)

Gel results:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 1st colony
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 1st colony
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 1st and 3rd colony
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 1st and 4th colony

We decided to keep two colonies for BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 and BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.

We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.

07/25/08

Glycerol stocks/miniprep for:
- BBa_J23100-BBa_B0030-BBa_C0012-BBa_B1006 (1)
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)

We sent these purified plasmids:
- BBa_B0030-BBa_I15009-BBa_BBa_B1006-BBa_R0082 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062 (3)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (1)
- BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006-BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (4)

to Primm for sequencing.

We put BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-BBa_R0079 plate at +4°C.

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	*to Primm for sequencing.		*to Primm for sequencing.
		+
		+	*We put BBa_B0030-BBa_C0061-BBa_B0030-BBa_C0078-BBa_B1006-'''BBa_R0079''' plate at +4°C.