Team:UNIPV-Pavia/Notebook/Week8

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!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]
!|[[Team:UNIPV-Pavia/Notebook/Week13|Week 13]]
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]
!|[[Team:UNIPV-Pavia/Notebook/Week14|Week 14]]
 +
|-
 +
!|[[Team:UNIPV-Pavia/Notebook/Week15|Week 15]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week16|Week 16]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week17|Week 17]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week18|Week 18]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week19|Week 19]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week20|Week 20]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week21|Week 21]]
 +
|-
 +
!|[[Team:UNIPV-Pavia/Notebook/Week22|Week 22]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week23|Week 23]]
 +
!|[[Team:UNIPV-Pavia/Notebook/Week24|Week 24]]
|}
|}
Line 50: Line 62:
**BBa_J23100-'''BBa_B0030'''-BBa_I15010 (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_E0040-'''BBa_B1006'''
-
**BBa_B0030-BBa_C0051-'''BBa_B1006'''
+
**BBa_B0030-BBa_C0051-'''BBa_B0030'''
**BBa_B0030-BBa_E1010-'''BBa_B1006'''
**BBa_B0030-BBa_E1010-'''BBa_B1006'''
{|
{|
-
|[[Image:pv_pcr_09_11_13_14_15.jpg|thumb|450px|left|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_B1006, '''BBa_B0030'''-BBa_E1010-BBa_B1006, blank]]
+
|[[Image:pv_pcr_09_11_13_14_15.jpg|thumb|650px|left|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]]
|}
|}
Line 60: Line 72:
**No true positives for BBa_J23100-'''BBa_B0030'''-BBa_C0012
**No true positives for BBa_J23100-'''BBa_B0030'''-BBa_C0012
**No true positives for BBa_J23100-'''BBa_B0030'''-BBa_I15010
**No true positives for BBa_J23100-'''BBa_B0030'''-BBa_I15010
-
**Pure true positives for BBa_B0030-BBa_C0051-'''BBa_B1006'''
+
**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:
 +
{|cellpadding="20px"
 +
|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...(@_@!)
 +
 
 +
<br><br>
 +
'''07/8/08'''
 +
<br>
 +
*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:
 +
{|cellpadding="20px"
 +
|BBa_B0030
 +
|BBa_I15010
 +
|BBa_B0030-BBa_E0040-'''BBa_B1006''' (1)
 +
|-
 +
|BBa_C0012
 +
|BBa_R0062
 +
|BBa_B0030-BBa_C0051-'''BBa_B0030'''
 +
|-
 +
|BBa_J23100-'''BBa_B0030'''
 +
|BBa_B0030-BBa_E1010-'''BBa_B1006''' (1)
 +
|}
 +
 
 +
*Miniprep for these parts.
 +
 
 +
*Plasmid digestion for:
 +
{|cellpadding="20px"
 +
|BBa_B0030 (S-P)
 +
|BBa_B0030-BBa_E0040-'''BBa_B1006''' (1) (E-X)
 +
|BBa_B0030-BBa_E1010-'''BBa_B1006''' (1) (E-X)
 +
|-
 +
|BBa_C0012 (X-P)
 +
|BBa_B0030-BBa_C0051-'''BBa_B0030''' (S-P)
 +
|BBa_J23100-'''BBa_B0030''' (E-S)
 +
|-
 +
|BBa_I15010 (X-P)
 +
|BBa_J23100-'''BBa_B0030'''-BBa_C0040 (E-S)
 +
|'''BBa_R0051'''-BBa_B0030-BBa_C0062 (E-S)
 +
|-
 +
|BBa_R0062 (E-X)
 +
|BBa_B0030-BBa_C0061-'''BBa_B1006''' (E-S)
 +
|}
 +
 
 +
*Gel run/cut/gel extraction for:
 +
{|cellpadding="20px"
 +
|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:
 +
{|cellpadding="20px"
 +
|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:
 +
#'''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'''
 +
#BBa_J23100-'''BBa_B0030'''-BBa_C0012 (again)
 +
#BBa_J23100-'''BBa_B0030'''-BBa_I15010 (again)
 +
#BBa_J23100-BBa_B0030-BBa_E0040-'''BBa_B1006''' (to test the part)
 +
#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.
 +
 
 +
<br><br>
 +
'''07/9/08'''
 +
<br>
 +
*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:
 +
{|cellpadding="20px"
 +
|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.
 +
 
 +
<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:
 +
#'''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'''
 +
#BBa_J23100-'''BBa_B0030'''-BBa_C0012
 +
#BBa_J23100-'''BBa_B0030'''-BBa_I15010
 +
 
 +
{|
 +
|[[Image:pv_falcon_colonypcr_9_7_08.jpg|thumb|300px|left|1 ml of infected LB + antibiotic for all the colonies we picked up to perform colony PCR]]
 +
||[[Image:pv_pcr_9_7_08.jpg|thumb|300px|left|Colony PCR]]
 +
|}
 +
 
 +
{|
 +
|[[Image:pv_pcr_9-11-18-20-22-23-24.jpg|thumb|850px|left|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:
 +
#'''BBa_B0030'''-BBa_I15009 1st colony was chosen.
 +
#BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' 5th colony was chosen.
 +
#BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' 2nd colony was chosen.
 +
#BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 4th colony was chosen.
 +
#BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' 3rd colont was chosen.
 +
#BBa_J23100-'''BBa_B0030'''-BBa_C0012 did not show true positive colonies.
 +
#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):
 +
 
 +
{|
 +
|[[Image:pv_G_R_10_7_08.png|thumb|700px|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.
 +
 
 +
{|
 +
|[[Image:pv_LB_tower_10_7_08.jpg|thumb|300px|left|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:
 +
{|cellpadding="20px"
 +
|BBa_C0012
 +
|'''BBa_B0030'''-BBa_I15009 (1)
 +
|-
 +
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (5)
 +
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (2)
 +
|-
 +
|BBa_J23100-'''BBa_B0030'''
 +
|BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 (4)
 +
|-
 +
|BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' (3)
 +
|}
 +
 
 +
*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.
 +
 
 +
<br><br>
 +
'''07/11/08'''
 +
<br>
 +
*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.
 +
 
 +
{|
 +
|[[Image:pv_pcr_17.png|thumb|600px|left|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:
 +
{|cellpadding="20px"
 +
|BBa_C0012
 +
|'''BBa_B0030'''-BBa_I15009 (1)
 +
|-
 +
|BBa_I15010
 +
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (5)
 +
|-
 +
|BBa_J23100-'''BBa_B0030'''
 +
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (2)
 +
|-
 +
|BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 (4)
 +
|BBa_B0030-BBa_C0061-BBa_B1006-'''BBa_R0062''' (3)
 +
|}
 +
 
 +
*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:
 +
{|cellpadding="20px"
 +
|BBa_C0012 (X-P)
 +
|'''BBa_B0030'''-BBa_I15009 (1) (E-S)
 +
|BBa_B1006 (we already had this plasmid at -20°C) (E-P)
 +
|-
 +
|BBa_J23100-'''BBa_B0030''' (E-S)
 +
|BBa_J23100-BBa_B0030-BBa_C0040-'''BBa_B1006''' (5) (E-S)
 +
|BBa_R0051-BBa_B0030-BBa_C0062-'''BBa_B1006''' (2) (E-S)
 +
|-
 +
|BBa_J23100-'''BBa_B0030''' (S-P)
 +
|BBa_R0040 (we already had this plasmid at -20°C) (E-X)
 +
|BBa_B0030-BBa_C0051-'''BBa_B0030'''-BBa_C0079 (4) (E-S)
 +
|}
 +
 
 +
*Gel run/cut/gel extraction for all these parts.
 +
 
 +
*(We already had BBa_B1006 (E-X) and BBa_R0062 (E-X))
 +
 
 +
*Ligation:
 +
#BBa_J23100-'''BBa_B0030''' (S-P) -BBa_C0012 (X-P) (1:2 ratio instead of 1:6)
 +
#'''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)
 +
#BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006-'''BBa_R0040'''
 +
#BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006-'''BBa_R0062'''
 +
#BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079-'''BBa_B1006'''
 +
#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.
 +
 
 +
<br><br>
 +
'''07/12/08'''
 +
<br>
 +
*Glycerol stocks for:
 +
{|cellpadding="20px"
 +
|BBa_R0010
 +
|BBa_R0079
 +
|BBa_B0030-BBa_C0078-'''BBa_B1006'''
 +
|}
 +
 
 +
*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!
 +
 
 +
<br>

Latest revision as of 21:26, 26 October 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 15 Week 16 Week 17 Week 18 Week 19 Week 20 Week 21
Week 22 Week 23 Week 24



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_C0012 BBa_R0062 BBa_B0030-BBa_C0051-BBa_B0030
BBa_J23100-BBa_B0030 BBa_B0030-BBa_E1010-BBa_B1006 (1)
  • Miniprep for these parts.
  • Plasmid digestion for:
BBa_B0030 (S-P) BBa_B0030-BBa_E0040-BBa_B1006 (1) (E-X) BBa_B0030-BBa_E1010-BBa_B1006 (1) (E-X)
BBa_C0012 (X-P) BBa_B0030-BBa_C0051-BBa_B0030 (S-P) BBa_J23100-BBa_B0030 (E-S)
BBa_I15010 (X-P) BBa_J23100-BBa_B0030-BBa_C0040 (E-S) BBa_R0051-BBa_B0030-BBa_C0062 (E-S)
BBa_R0062 (E-X) 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
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:
BBa_C0012 BBa_B0030-BBa_I15009 (1)
BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006 (5) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (2)
BBa_J23100-BBa_B0030 BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079 (4)
BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 (3)
  • 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:
BBa_C0012 BBa_B0030-BBa_I15009 (1)
BBa_I15010 BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006 (5)
BBa_J23100-BBa_B0030 BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (2)
BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079 (4) BBa_B0030-BBa_C0061-BBa_B1006-BBa_R0062 (3)
  • 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:
BBa_C0012 (X-P) BBa_B0030-BBa_I15009 (1) (E-S) BBa_B1006 (we already had this plasmid at -20°C) (E-P)
BBa_J23100-BBa_B0030 (E-S) BBa_J23100-BBa_B0030-BBa_C0040-BBa_B1006 (5) (E-S) BBa_R0051-BBa_B0030-BBa_C0062-BBa_B1006 (2) (E-S)
BBa_J23100-BBa_B0030 (S-P) BBa_R0040 (we already had this plasmid at -20°C) (E-X) BBa_B0030-BBa_C0051-BBa_B0030-BBa_C0079 (4) (E-S)
  • 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:
BBa_R0010 BBa_R0079 BBa_B0030-BBa_C0078-BBa_B1006
  • 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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