Team:UNIPV-Pavia/Notebook/Week10
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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]] | ||
|} | |} | ||
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'''07/21/08''' | '''07/21/08''' | ||
<br> | <br> | ||
- | *Colony PCR for | + | *Colony PCR for B0030-C0078-B1006-'''R0079''': 6 colonies from single colonies plate. |
{| | {| | ||
- | |[[Image:pv_colonypcr_25_single_col.jpg|thumb|300px|left| | + | |[[Image:pv_colonypcr_25_single_col.jpg|thumb|300px|left|B0030-C0078-B1006-'''R0079''']] |
|} | |} | ||
- | *Gel results: all the 6 picked colonies were true positive! we chose the 5th | + | *Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture. |
- | *We also infected 9 ml LB + Amp with 30 µl of | + | *We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-'''R0040''' and '''B0030'''-C0061 glycerol stocks to grow two overnight cultures. |
*Ligation: | *Ligation: | ||
- | ** | + | **J23100-B0030-C0012-'''B1006''' (1:2 ratio, 40 ng of vector) |
- | ** | + | **B0030-I15009-B1006-'''R0082''' (30 ng of vector) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 |
*We incubated ligations at 16°C overnight. | *We incubated ligations at 16°C overnight. | ||
Line 71: | Line 83: | ||
*Glycerol stocks/miniprep for: | *Glycerol stocks/miniprep for: | ||
{|cellpadding="20px" | {|cellpadding="20px" | ||
- | | | + | |J23100-B0030-C0040-B1006-'''R0040''' |
- | |''' | + | |'''B0030'''-C0061 |
|- | |- | ||
- | | | + | |B0030-C0078-B1006-'''R0079''' |
|} | |} | ||
- | *We sent | + | *We sent J23100-B0030-C0040-B1006-'''R0040''' and B0030-C0078-B1006-'''R0079''' purified plasmids to Primm for sequencing. |
*Plasmid digestion for: | *Plasmid digestion for: | ||
{|cellpadding="20px" | {|cellpadding="20px" | ||
- | |''' | + | |'''B0030'''-C0061 (E-S) |
- | | | + | |B0030-C0078-B1006-'''R0079''' (E-X) |
|} | |} | ||
Line 92: | Line 104: | ||
*Ligation plates showed colonies!We put ligation plates at +4°C. | *Ligation plates showed colonies!We put ligation plates at +4°C. | ||
- | *Ligation for | + | *Ligation for B0030-C0061-B0030-C0078-B1006-'''R0079'''. |
*We incubated ligation at 16°C overnight. | *We incubated ligation at 16°C overnight. | ||
Line 99: | Line 111: | ||
'''07/24/08''' | '''07/24/08''' | ||
<br> | <br> | ||
- | *Transformation (1 µl) for | + | *Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-'''R0079''' ligation. We plated transformed bacteria and incubated plate at 37°C overnight. |
*Colony PCR for: | *Colony PCR for: | ||
- | ** | + | **J23100-B0030-C0012-'''B1006''' |
- | ** | + | **B0030-I15009-B1006-'''R0082''' |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 |
- | *(We performed one PCR for | + | *(We performed one PCR for J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''' with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation). |
{|cellpadding="1px" | {|cellpadding="1px" | ||
- | |[[Image:pv_pcr_parallel1.jpg|thumb|300px|left|Thermal cycler working on | + | |[[Image:pv_pcr_parallel1.jpg|thumb|300px|left|Thermal cycler working on J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''' colonies: 2 min elongation]] |
- | |[[Image:pv_pcr_parallel2.jpg|thumb|300px|left|Thermal cycler working on | + | |[[Image:pv_pcr_parallel2.jpg|thumb|300px|left|Thermal cycler working on B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 colonies: 3 min elongation]] |
|} | |} | ||
- | *Electrophoresis (1.2% agarose) for | + | *Electrophoresis (1.2% agarose) for J23100-B0030-C0012-'''B1006''' and B0030-I15009-B1006-'''R0082''': |
{| | {| | ||
- | |[[Image:pv_colonypcr_19_32.jpg|thumb|300px|left|Marker 1Kb, blank, | + | |[[Image:pv_colonypcr_19_32.jpg|thumb|300px|left|Marker 1Kb, blank, J23100-B0030-C0012-'''B1006''' (7 lanes), B0030-I15009-B1006-'''R0082''' (6 lanes)]] |
|} | |} | ||
- | *Electrophoresis (1% agarose) for | + | *Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006: |
{| | {| | ||
- | |[[Image:pv_colonypcr_34_35.jpg|thumb|300px|left|Marker 1Kb, | + | |[[Image:pv_colonypcr_34_35.jpg|thumb|300px|left|Marker 1Kb, B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (7 lanes), B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (7 lanes)]] |
|} | |} | ||
*Gel results: | *Gel results: | ||
- | ** | + | **J23100-B0030-C0012-'''B1006''' 1st colony |
- | ** | + | **B0030-I15009-B1006-'''R0082''' 1st colony |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 1st and 4th colony |
+ | |||
+ | *We decided to keep two colonies for B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct. | ||
- | * | + | *The fifth colony of B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies. |
*We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures. | *We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures. | ||
Line 140: | Line 154: | ||
<br> | <br> | ||
*Glycerol stocks/miniprep for: | *Glycerol stocks/miniprep for: | ||
- | ** | + | **J23100-B0030-C0012-'''B1006''' (1) |
- | ** | + | **B0030-I15009-B1006-'''R0082''' (1) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4) |
*We sent these purified plasmids: | *We sent these purified plasmids: | ||
- | ** | + | **B0030-I15009-B1006-'''R0082''' (1) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (1) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006-R0062 (3) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (1) |
- | ** | + | **B0030-C0051-B0030-C0079-'''B1006'''-R0051-B0030-C0062-B1006 (4) |
*to Primm for sequencing. | *to Primm for sequencing. | ||
- | *We put | + | *We put B0030-C0061-B0030-C0078-B1006-'''R0079''' plate at +4°C. |
Latest revision as of 21:26, 26 October 2008
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Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |
Week 22 | Week 23 | Week 24 |
Week 10: 07/21/08 - 07/25/08
07/21/08
- Colony PCR for B0030-C0078-B1006-R0079: 6 colonies from single colonies plate.
- Gel results: all the 6 picked colonies were true positive! we chose the 5th colony to grow a 9 ml overnight culture.
- We also infected 9 ml LB + Amp with 30 µl of J23100-B0030-C0040-B1006-R0040 and B0030-C0061 glycerol stocks to grow two overnight cultures.
- Ligation:
- J23100-B0030-C0012-B1006 (1:2 ratio, 40 ng of vector)
- B0030-I15009-B1006-R0082 (30 ng of vector)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006
- We incubated ligations at 16°C overnight.
07/22/08
- Transformation for the 4 overnight ligations (1 µl). We plated transformed bacteria and incubated plates at 37°C overnight.
- Glycerol stocks/miniprep for:
J23100-B0030-C0040-B1006-R0040 | B0030-C0061 |
B0030-C0078-B1006-R0079 |
- We sent J23100-B0030-C0040-B1006-R0040 and B0030-C0078-B1006-R0079 purified plasmids to Primm for sequencing.
- Plasmid digestion for:
B0030-C0061 (E-S) | B0030-C0078-B1006-R0079 (E-X) |
- Gel run/cut/gel extraction.
07/23/08
- Ligation plates showed colonies!We put ligation plates at +4°C.
- Ligation for B0030-C0061-B0030-C0078-B1006-R0079.
- We incubated ligation at 16°C overnight.
07/24/08
- Transformation (1 µl) for B0030-C0061-B0030-C0078-B1006-R0079 ligation. We plated transformed bacteria and incubated plate at 37°C overnight.
- Colony PCR for:
- J23100-B0030-C0012-B1006
- B0030-I15009-B1006-R0082
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006
- (We performed one PCR for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082 with 2 min elongation and another PCR for the remaining two ligations with 3 min elongation).
- Electrophoresis (1.2% agarose) for J23100-B0030-C0012-B1006 and B0030-I15009-B1006-R0082:
- Electrophoresis (1% agarose) for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006:
- Gel results:
- J23100-B0030-C0012-B1006 1st colony
- B0030-I15009-B1006-R0082 1st colony
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 1st and 3rd colony
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 1st and 4th colony
- We decided to keep two colonies for B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 and B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 ligations because they are important parts for our project and we wanted to be sure they were correct.
- The fifth colony of B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 showed an unexpected length...(@_@?) We decided to ignore it. Sequencing results will tell us if we made mistakes in assemblies.
- We infected 9 ml LB + Amp with chosen colonies to grow 6 overnight cultures.
07/25/08
- Glycerol stocks/miniprep for:
- J23100-B0030-C0012-B1006 (1)
- B0030-I15009-B1006-R0082 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
- We sent these purified plasmids:
- B0030-I15009-B1006-R0082 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006-R0062 (3)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (1)
- B0030-C0051-B0030-C0079-B1006-R0051-B0030-C0062-B1006 (4)
- to Primm for sequencing.
- We put B0030-C0061-B0030-C0078-B1006-R0079 plate at +4°C.