Team:UNIPV-Pavia/Notebook/Week8

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Revision as of 09:23, 21 July 2008

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Notebook

Week 8: 07/7/08 - 07/12/08

07/7/08

• Colony PCR (5 colonies for each plate) for:
  • BBa_J23100-BBa_B0030-BBa_C0012 (for the second time, we hoped to find true positive colonies)
  • BBa_J23100-BBa_B0030-BBa_I15010 (for the second time, we hoped to find true positive colonies)
  • BBa_B0030-BBa_E0040-BBa_B1006
  • BBa_B0030-BBa_C0051-BBa_B0030
  • BBa_B0030-BBa_E1010-BBa_B1006

Colony PCR: Marker (1), empty (2) BBa_J23100-BBa_B0030-BBa_C0012, BBa_J23100-BBa_B0030-BBa_I15010, BBa_B0030-BBa_E0040-BBa_B1006, BBa_B0030-BBa_C0051-BBa_B0030, BBa_B0030-BBa_E1010-BBa_B1006, blank

• Gel results:
  • No true positives for BBa_J23100-BBa_B0030-BBa_C0012
  • No true positives for BBa_J23100-BBa_B0030-BBa_I15010
  • Non pure true positives for BBa_B0030-BBa_E0040-BBa_B1006
  • Pure true positives for BBa_B0030-BBa_C0051-BBa_B0030
  • Non pure true positives for BBa_B0030-BBa_E1010-BBa_B1006

• We chose to keep the first colony for all the 3 working ligation plates.

• NOTE: BBa_B0030-BBa_E0040-BBa_B1006 and BBa_B0030-BBa_E1010-BBa_B1006 are final parts; we decided to sequence these 2 parts even if gel showed a weak contamination: sequencing results will tell us if there are false positive plasmids in those colonies or if the contamination was only a PCR contamination.

• We infected 9 ml LB + suitable antibiotic with 30 µl of these glycerol stocks:

• We incubated the 8 cultures at 37°C, 220 rpm overnight.

• Tomorrow we will be ready to perform NINE LIGATIONS...(@_@!)

07/8/08

• We received sequencing results for BBa_B0030-BBa_C0078: sequence showed an extra DNA fragment between BBa_C0078 and Suffix...We decided to ignore it because there was a stop codon before that fragment.

• Glycerol stocks for:

• Miniprep for these parts.

• Plasmid digestion for:

• Gel run/cut/gel extraction for:

• DNA precipitation with sodium acetate for:

• (We already had BBa_I15009(X-P) and BBa_B1006(E-X))

• Ligations:

1. BBa_B0030-BBa_I15009
2. BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006
3. BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
4. BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079
5. BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062
6. BBa_J23100-BBa_B0030-BBa_C0012 (again)
7. BBa_J23100-BBa_B0030-BBa_I15010 (again)
8. BBa_J23100-BBa_B0030-BBa_E0040-BBa_B1006 (to test the part)
9. BBa_J23100-BBa_B0030-BBa_E1010-BBa_B1006 (to test the part)

• We incubated ligations at 16°C overnight.

• We infected 9 ml LB + Amp with 30 µl of BBa_B1006 and BBa_B0030-BBa_C0078 glycerol stocks. We incubated the 2 cultures at 37°C, 220 rpm ovenight.

07/9/08

• We transformed the 9 ligations (2 µl) and plated transformed bacteria. We incubated plates at 37°C ovenight.

• Glycerol stocks for BBa_B1006 and BBa_B0030-BBa_C0078.

• Miniprep for BBa_B1006 and BBa_B0030-BBa_C0078.

• Plasmid digestion:

• Gel run/cut/gel extraction for BBa_B0030-BBa_C0078 (E-S).

• DNA precipitation with sodium acetate for BBa_B1006 (E-X).

• Ligation: BBa_B0030-BBa_C0078-BBa_B1006

• We incubated ligation at 16°C overnight.

07/10/08

• We transformed BBa_B0030-BBa_C0078-BBa_B1006 ligation (2 µl) and plated transformed bacteria. We incubated the plate at 37°C ovenight.

• All the ligation plates showed colonies! There were carpets, but we could pick up some single colonies for colony PCR.

• Colony PCR for:

1. BBa_B0030-BBa_I15009
2. BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006
3. BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006
4. BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079
5. BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062
6. BBa_J23100-BBa_B0030-BBa_C0012
7. BBa_J23100-BBa_B0030-BBa_I15010

1 ml of infected LB + antibiotic for all the colonies we picked up to perform colony PCR

Colony PCR

Colony PCR: Marker (1), BBa_J23100-BBa_B0030-BBa_C0012, BBa_J23100-BBa_B0030-BBa_I15010, BBa_B0030-BBa_I15009, BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006, BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006, BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079, BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062

• Gel results:

1. BBa_B0030-BBa_I15009 1st colony was chosen.
2. BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006 5th colony was chosen.
3. BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 2nd colony was chosen.
4. BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079 4th colony was chosen.
5. BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 3rd colont was chosen.
6. BBa_J23100-BBa_B0030-BBa_C0012 did not show true positive colonies.
7. BBa_J23100-BBa_B0030-BBa_I15010 did not show true positive colonies.

• We streaked BBa_J23100-BBa_B0030-BBa_E0040-BBa_B1006 and BBa_J23100-BBa_B0030-BBa_E1010-BBa_B1006 with a top to test the parts (NOTE: we could see some red colonies on BBa_J23100-BBa_B0030-BBa_E1010-BBa_B1006 plate!these colonies correspond to bacteria with correctly ligated plasmid, while normal color colonies correspond to bacteria with BBa_B0030-BBa_E1010-BBa_B1006 plasmid, without constitutive promoter).
  • We infected 100 µl LB + Amp with the tips and incubated the culture at 37°C, 220 rpm for 2 hours.
  • Then we watched green, blue and red fluorescence channels at microscope (50 µl of each culture):

3 acquisitions for red cells; 3 acquisition for green cells; one acquisition for DAPI channel (blue) for green cells to check for impurities (DAPI channel acquisition for red cells was not saved, but it didn't show any blue area)

• LB preparation: 0.5 l LB + Amp for plates.

Prepared LB plates tower

• BBa_R0010 resuspension from Registry of Standard Parts.

• We transformed (4 µl) BBa_R0010 and plated transformed bacteria. We incubated BBa_R0010 plate at 37°C overnight.

• We infected 9 ml LB + suitable antibiotic with 30 µl of these glycerol stocks:

• We also picked up one colony from BBa_I15010 plate with a tip and infected 9 ml LB + Kan. We incubated the 8 cultures at 37°C, 220 rpm overnight.

07/11/08

• BBa_R0010 plate showed 2 colonies. We picked up one colony and infected 9 ml LB + Amp to grow an overnight culture.

• BBa_B0030-BBa_C0078-BBa_B1006 plate showed many colonies. We used 7 colonies to perform colony PCR.

7 colonies for BBa_B0030-BBa_C0078-BBa_B1006

• Gel results: all the 7 colonies contained ligated plasmid; we chose the 5th colony to grow a 9 ml overnigth culture because 5th it showed no impurities.

• Glycerol stocks for:

• Miniprep for these 8 parts.

• We sent BBa_I15010 and BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 (3) to Primm for sequencing.

• Plasmid digestion for:

• Gel run/cut/gel extraction for all these parts.

• (We already had BBa_B1006 (E-X) and BBa_R0062 (E-X))

• Ligation:

1. BBa_J23100-BBa_B0030 (S-P) -BBa_C0012 (X-P) (1:2 ratio instead of 1:6)
2. BBa_B1006 (E-P) + BBa_J23100-BBa_B0030 (E-S, 1:4) -BBa_C0012 (X-P, 1:2) (we decided to try this double ligation)
3. BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-BBa_R0040
4. BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-BBa_R0062
5. BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-BBa_B1006
6. BBa_B0030-BBa_I15009-BBa_B1006

• We incubated ligations at 16°C overnight.

• We infected 9 ml LB + Amp with 30 µl of BBa_R0079 glycerol stock.

07/12/08

• Glycerol stocks for:

• Miniprep for these 3 parts.

• We put these 3 purified plasmids at -20°C.

• We also put the 6 ovenight ligations at -20°C. Next week they will be transformed!

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