Team:Warsaw/Calendar-Main/11 July 2009
From 2009.igem.org
(Difference between revisions)
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*Isolation of fragment of the correct lenght(˜2200bp)from the gel was performed with the [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf A&A "Gel-out" kit]. | *Isolation of fragment of the correct lenght(˜2200bp)from the gel was performed with the [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf A&A "Gel-out" kit]. | ||
+ | |||
+ | |||
+ | ===Cloning the p53 coding sequence=== | ||
+ | |||
+ | '''Marcin''' | ||
+ | |||
+ | Task 1: | ||
+ | *Breed bacteria to isolate plasmid containing p53 coding sequence | ||
+ | Methods: | ||
+ | # Prepare LB medium with kanamycin | ||
+ | # Add 3.5 ml of the medium to the probes | ||
+ | # Add one bacterial colony to each probe | ||
+ | # Breed the bacteria about 7 hours | ||
+ | Task 2: | ||
+ | *Isolate the plasmids from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis | ||
+ | Methods: | ||
+ | *Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here] | ||
+ | *After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel | ||
+ | *Electrophoresis condition: | ||
+ | |||
+ | voltage - 70V | ||
+ | |||
+ | time - 30 min | ||
+ | |||
+ | *Next the gel was photographed: | ||
+ | |||
+ | |||
+ | [[image:P53_izolacja_plazmidu_11_07_09.png |thumb|450px|center| plasmid samples on the gel]] | ||
+ | |||
+ | |||
+ | |||
=== <div style="text-align: center;">Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2 <div>=== | === <div style="text-align: center;">Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2 <div>=== |
Revision as of 10:23, 12 July 2009
Contents |
Gel out phoP/phoQ
Kama
Tasks:
- Isolation of fragment of the correct lenght(˜2200bp)from the gel was performed with the A&A "Gel-out" kit.
Cloning the p53 coding sequence
Marcin
Task 1:
- Breed bacteria to isolate plasmid containing p53 coding sequence
Methods:
- Prepare LB medium with kanamycin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- Breed the bacteria about 7 hours
Task 2:
- Isolate the plasmids from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis
Methods:
- Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
- After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel
- Electrophoresis condition:
voltage - 70V
time - 30 min
- Next the gel was photographed:
Construction of K177012 operon1_part2
Ania
Tasks:
- Transformation of chemocompetent strain of E. with BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid taken out of the distribution 200 Kit Plate 1 well 2O.
- Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:
BBa_R0051 - promoter (lambda cI regulated);
BBa_B0032 - RBS.3 (medium)
- Digestion to confirm plasmid extraction. DNA that should contain R0051 on pSB1A2 plasmid was digested with PvuI and HindII. Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer , 0.3µl of each enzyme and water added to obtain 20µl total volume.
DNA sample
restriction enzymes
expected fragments [bp]
R0051(pcI) on pSB1A2
PvuI HindII
695, 1447
Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek
Tasks:
- Alkaline lysis of bacterial cultures to obtain plasmids containing BBa_R0010 - lacI regulated promoter and BBa_R0080 - AraC regulated promoter bricks
- Digestion to confirm plasmid extraction.
Methods:
- 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing placI or pAraC bricks on pSB1A2 plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.
- DNA that should contain pAraC on pSB1A2 plasmid was digested with BamHI and PvuI, placI on pSB1A2 was digested with IPvuI and PvuII . Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer, 0.3µl of each enzyme and water added to obtain 20µl total volume. All mixes were incubated for 2h at 37°C.
DNA sample
restriction enzymes
expected fragments [bp]
placI on pSB1A2
PvuI, PvuII
728, 1351
pAraC on pSB1A2
BamHI, PvuI
782, 1446
Results:
Franek lub Monika opisze minilizy dla Moniki
April
M T W T F S S
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May
M T W T F S S
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June
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July
M T W T F S S
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August
M T W T F S S
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September
M T W T F S S
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October
M T W T F S S
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Ania
Tasks:
- Transformation of chemocompetent strain of E. with BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid taken out of the distribution 200 Kit Plate 1 well 2O.
- Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:
BBa_R0051 - promoter (lambda cI regulated); BBa_B0032 - RBS.3 (medium)
- Digestion to confirm plasmid extraction. DNA that should contain R0051 on pSB1A2 plasmid was digested with PvuI and HindII. Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer , 0.3µl of each enzyme and water added to obtain 20µl total volume.
DNA sample | restriction enzymes | expected fragments [bp] |
---|---|---|
R0051(pcI) on pSB1A2 | PvuI HindII | 695, 1447 |
Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek
Tasks:
- Alkaline lysis of bacterial cultures to obtain plasmids containing BBa_R0010 - lacI regulated promoter and BBa_R0080 - AraC regulated promoter bricks
- Digestion to confirm plasmid extraction.
Methods:
- 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing placI or pAraC bricks on pSB1A2 plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.
- DNA that should contain pAraC on pSB1A2 plasmid was digested with BamHI and PvuI, placI on pSB1A2 was digested with IPvuI and PvuII . Digestion mix contained 5µl of extracted DNA, 2µl of Fermentas BamHI buffer, 0.3µl of each enzyme and water added to obtain 20µl total volume. All mixes were incubated for 2h at 37°C.
DNA sample | restriction enzymes | expected fragments [bp] |
---|---|---|
placI on pSB1A2 | PvuI, PvuII | 728, 1351 |
pAraC on pSB1A2 | BamHI, PvuI | 782, 1446 |
Results:
Franek lub Monika opisze minilizy dla Moniki
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