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Week 11: 07/28/08 - 08/1/08
- Colony PCR for B0030-C0061-B0030-C0078-B1006-R0079 plate: 6 colonies.
- Gel results: all colonies were true positives! we decided to keep 1st colony to grow a 9 ml overnight culture.
- Plasmid digestion for:
|J23100-B0030-C0012-B1006 (E-S)||R0010 (E-X)|
- Gel run/cut/gel extraction.
- Ligation: J23100-B0030-C0012-B1006-R0010
- We incubated ligation at 16°C overnight.
- We infected 9 ml LB + Amp with 30 µl of these glycerol stocks:
|B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)||B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)|
- LB + Amp preparation.
- Glycerol stocks/miniprep for the 7 overnight cultures. Unfortunately we had low yield...
- DNA precipitation with sodium acetate for the 7 purified plasmids to concetrate DNA in a final volume of 10 µl.
- We transformed J23100-B0030-C0012-B1006-R0010 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
- We received sequencing results for:
- J23100-B0030-C0040-B1006-R0040: sequence was not correct...we ligated the wrong part in the last ligation step of this composite part. We decided to re-ligate J23100-B0030-C0040-B1006 and R0040.
- B0030-C0078-B1006-R0079: apart from an extra dna fragment at the end of C0078 (already noticed in previous sequencing), sequence was correct!
- Plasmid digestion for:
|B0030-BBa_E0040-BBa_B1006 (X-P)||B0030-BBa_C0061-BBa_B1006-BBa_R0062 (S-P)|
|B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1) (S-P)||B0030-C0061-B0030-C0079-B1006-R0079 (1) (S-P)|
|B0030-E1010-B1006 (X-P)||B0030-C0078-B1006-R0079 (S-P)|
- Gel run/cut/gel extraction. DNA quantification with NanoDrop showed that all cut parts were enough for the four ligations!
- We incubated ligations at 16°C overnight. These ligations are test assemblies, in fact reporter protein generators are assembled downstream of promoters.
- We infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006 and R0040 glycerol stocks. We incubated cultures at 37°C, 220 rpm overnight.
- We transformed overnight ligations. We plated transformed bacteria and incubated plates at 37°C overnight.
- Glycerol stocks/miniprep for J23100-B0030-C0040-B1006 and R0040. Next week we will be ready to re-perform the unlucky ligation between these two parts;)
- Colony PCR for J23100-B0030-C0012-B1006-R0010 (the plate was stored at +4°C): we picked up 12 colonies.
- Gel results: we couldn't see any band from gel...maybe we made a mistake in PCR mix...Next week we will re-perform this colony PCR.
- Sequencing results: we had a point mutation (C->T) in C0062 coding sequence, in B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 composite parts...This mutation determines a Ser->Phe change in DNA binding domain of luxR gene. We decided to test mutated part anyway, to understand if protein function is actually compromised. In the meanwhile, we decided to sequence all parts with C0062 coding sequence to find the step in which mutation occurred and the re-perform ligations.
- We put:
- ligation plates at +4°C. Next week we will perform colony PCR on these plates.