Team:UNIPV-Pavia/Notebook/Week8

From 2008.igem.org

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'''07/9/08'''
'''07/9/08'''
<br>
<br>
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*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.
*Glycerol stocks for BBa_B1006 and '''BBa_B0030'''-BBa_C0078.
*Miniprep for BBa_B1006 and '''BBa_B0030'''-BBa_C0078.
*Miniprep for BBa_B1006 and '''BBa_B0030'''-BBa_C0078.
 +
 +
*Plasmid digestion:
 +
{|cellpadding="20px"
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|BBa_B1006 (E-X)
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|'''BBa_B0030'''-BBa_C0078 (E-S)
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|}
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*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'''
 +
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*We incubated ligation at 16°C overnight.
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 +
<br><br>
 +
'''07/10/08'''
 +
<br>
 +
*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

Revision as of 11:28, 20 July 2008


Home.jpg Home Unipv logo.jpg The Team And.jpg The Project Safety.jpg Biological Safety Dna.png Parts Submitted to the Registry
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
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