Team:UNIPV-Pavia/Notebook/Week6

Week 6: 06/23/08 - 06/27/08
06/23/08
 * Colony PCR for BBa_B0030-BBa_C0061 and BBa_B0030 (no Ant.Phosph.)-BBa_C0061: 10 colonies for every plate.


 * We ran PCR results: some colonies showed both ligated plasmid (heavier band) and false positive plasmid, because it was difficult to pick up single colonies from bacteria carpet. Ligation with Antarctic Phosphatase showed non-pure colonies and 3 false positives. Ligation without Antarctic Phosphatase showed 3 non-pure colonies and 7 pure colonies.


 * We decided to avoid Antarctic Phosphatase treatment in next ligations to save time.


 * We also decided to cut up to 1 µg of vector, to reduce background noise.


 * We choose 4th and 7th colonies to grow 9 ml cultures overnight.


 * We also infected 9 ml LB + Amp with 30 µl of:
 * glycerol stocks. We incubated all the 6 cultures at 37°C, 220 rpm.


 * We transformed 10 µl of the remaining ligations:
 * BBa_B0030-BBa_E0040
 * BBa_B0030-BBa_E1010
 * BBa_B0030-BBa_C0051


 * Wiki updating: Project section.

06/24/08
 * All the 3 ligation plates showed carpets. We decided to streak plates to produce single colonies plates. We incubated the 3 single colonies plates at 37°C overnight.


 * Glycerol stocks for:


 * Miniprep for these 6 plasmids.


 * Plasmid digestion:


 * DNA precipitation with sodium acetate for BBa_J23100 (S-P) and BBa_B0030 (S-P).


 * Gel run for BBa_E0240 (X-P) and BBa_C0078 (X-P)


 * Gel extraction.


 * Ligation:
 * BBa_J23100-BBa_E0240
 * BBa_B0030-BBa_C0078

06/25/08
 * We transformed 5 µl of the two ligations.


 * We also transformed 0.5 µl of BBa_B0030(S-P) and BBa_J23100(S-P) plasmids to estimate background noise.


 * Colony PCR (6 colonies for each plate) for:
 * BBa_B0030-BBa_E0040
 * BBa_B0030-BBa_E1010
 * BBa_B0030-BBa_C0051


 * The gel showed many working ligations! we choose the 1st colony for BBa_B0030-BBa_E0040, the 6th colony for BBa_B0030-BBa_C0051 and the 2nd colony for BBa_B0030-BBa_E1010 to grow 9 ml LB + Amp overnight cultures.

06/26/08
 * Glycerol stocks for:
 * BBa_B0030-BBa_E0040 (1)
 * BBa_B0030-BBa_E1010 (6)
 * BBa_B0030-BBa_C0051 (2)


 * Miniprep for these 3 ligations. We sent purified plasmids to Primm for sequencing.


 * BBa_J23100-BBa_E0240 and BBa_B0030-BBa_C0078 plates showed a carpet.


 * We prepared single colonies plates for BBa_J23100-BBa_E0240 and BBa_B0030-BBa_C0078.


 * NOTE: BBa_J23100-BBa_E0240 plate showed red colonies (false positives) and normal color colonies (ligations).


 * We tested fluorescence for BBa_J23100-BBa_E0240: we streaked the plate and infected 100 µl of LB + Amp. We incubated this culture for 2 hours at 37°C, 220 rpm. Then we watched green, blue and red fluorescence channels at microscope. Some cells expressed GFP (cells with ligated plasmid) and some cells expressed RFP (false positives).

06/27/08 We put BBa_J23100-BBa_E0240 and BBa_B0030-BBa_C0078 single colonies plates at +4°C. Next week we will perform colony PCR for these two ligations.