Team:UNIPV-Pavia/Notebook/Week6

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Notebook

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.

Colony PCR for BBa_B0030-BBa_C0061: Antarctic Phosphatase results (top) and non-Antarctic Phosphatase results (bottom)

• 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

Colony PCR for BBa_B0030-BBa_E0040, BBa_B0030-BBa_E0051 and BBa_B0030-BBa_E1010: Marker and 6 colonies for each ligation

• 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).

Cells with ligated plasmid	False positive cells
Blue channel (to be sure that our glowing cells were not impurities)	Merge: green, red and blue channels

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.

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