Team:UNIPV-Pavia/Notebook/Week8

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*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 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.
**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:
 +
 +
{|
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|[[Image:pv_G_R_10_7_08.jpg|thumb|500px|left|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.
*LB preparation: 0.5 l LB + Amp for plates.

Revision as of 12:37, 20 July 2008


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Laptop.jpg Dry Lab Pipette.jpg Wet Lab Math.gif Modeling Note.jpg Protocols Notebook.gif Activity Notebook



Notebook



Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7
Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14



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:
BBa_B0030 BBa_I15010 BBa_B0030-BBa_E0040-BBa_B1006 (1) BBa_B0030-BBa_E1010-BBa_B1006 (1)
BBa_C0012 BBa_R0062 BBa_B0030-BBa_C0051-BBa_B0030 BBa_J23100-BBa_B0030
  • 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:
BBa_B0030 BBa_I15010 BBa_B0030-BBa_E0040-BBa_B1006 (1) BBa_B0030-BBa_E1010-BBa_B1006 (1)
BBa_C0012 BBa_R0062 BBa_B0030-BBa_C0051-BBa_B0030 BBa_J23100-BBa_B0030
  • Miniprep for these parts.
  • Plasmid digestion for:
BBa_B0030 (S-P) BBa_I15010 (X-P) BBa_B0030-BBa_E0040-BBa_B1006 (1) (E-X) BBa_B0030-BBa_E1010-BBa_B1006 (1) (E-X)
BBa_C0012 (X-P) BBa_R0062 (E-X) BBa_B0030-BBa_C0051-BBa_B0030 (S-P) BBa_J23100-BBa_B0030 (E-S)
BBa_J23100-BBa_B0030-BBa_C0040 (E-S) BBa_R0051-BBa_B0030-BBa_C0062 (E-S) BBa_B0030-BBa_C0061-BBa_B1006 (E-S)
  • Gel run/cut/gel extraction for:
BBa_I15010 (X-P) BBa_J23100-BBa_B0030 (E-S) BBa_J23100-BBa_B0030-BBa_C0040 (E-S)
BBa_C0012 (X-P) BBa_R0051-BBa_B0030-BBa_C0062 (E-S) BBa_B0030-BBa_C0061-BBa_B1006 (E-S)
  • DNA precipitation with sodium acetate for:
BBa_B0030 (S-P) BBa_B0030-BBa_E0040-BBa_B1006 (1) (E-X) BBa_B0030-BBa_E1010-BBa_B1006 (1) (E-X)
BBa_R0062 (E-X) BBa_B0030-BBa_C0051-BBa_B0030 (S-P)
  • (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:
BBa_B1006 (E-X) BBa_B0030-BBa_C0078 (E-S)
  • 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
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:
File:Pv G R 10 7 08.jpg
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.
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