Team:Warsaw/Calendar-Main/11 July 2009
From 2009.igem.org
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- | ==== | + | ===<div style="text-align: center;">Insertion of the pho gene into the pKSII+ plasmid</div> === |
- | *Isolation of fragment of the correct lenght(& | + | '''Kama''' |
+ | __NOEDITSECTION__ | ||
+ | Tasks: | ||
+ | |||
+ | *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]. | ||
+ | |||
+ | https://static.igem.org/mediawiki/2009/0/08/2009_07_12_pho_after_gelout_opisana.JPG | ||
+ | |||
+ | *purified inserts of biggest concentration were taken to next steps of cloning (marked with an arrow, ~30ng/μl) | ||
+ | |||
+ | <h3>Cloning of p53 coding sequence</h3> | ||
+ | __NOEDITSECTION__ | ||
+ | <h4>Marcin</h4> | ||
+ | |||
+ | 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>=== | ||
+ | __NOEDITSECTION__ | ||
+ | '''Ania''' | ||
- | |||
Tasks: | Tasks: | ||
Line 15: | Line 56: | ||
[http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032] - RBS.3 (medium) | [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 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 | + | * 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. |
{| class="wikitable" style="text-align:center; width:500px; height:90px; border-collapse: collapse; border: 1px dashed blue; margin: 1em auto 1em auto" border="1" | {| class="wikitable" style="text-align:center; width:500px; height:90px; border-collapse: collapse; border: 1px dashed blue; margin: 1em auto 1em auto" border="1" | ||
|- | |- | ||
! DNA sample | ! DNA sample | ||
! restriction enzymes | ! restriction enzymes | ||
- | ! expected fragments | + | ! expected fragments [bp] |
|- | |- | ||
| R0051(pcI) on pSB1A2 | | R0051(pcI) on pSB1A2 | ||
| PvuI HindII | | PvuI HindII | ||
- | | | + | | 695, 1447 |
- | |} | + | |} |
+ | [[image:110709_trawienia_promororow_Ania_Franek.jpg]] | ||
+ | <br> | ||
+ | * obtained fragments match expected | ||
+ | <!-- TU EDYTUJE FRANEK, NIE RUSZ! --> | ||
- | ==== | + | ===<div style="text-align: center;">Creating devices to test promoters in <em>E. coli</em> strains (devices [http://partsregistry.org/Part:BBa_K177024 <span style="color: black;">BBa_K177024</span>] and [http://partsregistry.org/Part:BBa_K177025 <span style="color: black;">BBa_K177025</span>])</div>=== |
- | + | ||
- | [http://partsregistry.org/ | + | |
- | [http://partsregistry.org/ | + | |
- | * Digestion to confirm plasmid extraction. | + | '''Franek''' |
+ | __NOEDITSECTION__ | ||
+ | <br> | ||
+ | Tasks: | ||
+ | |||
+ | * Alkaline lysis of bacterial cultures to obtain plasmids containing [http://partsregistry.org/Part:BBa_R0010 <span style="color: black;">BBa_R0010 - lacI regulated promoter</span>] and [http://partsregistry.org/Part:BBa_R0080 <span style="color: black;">BBa_R0080 - AraC regulated promoter</span>] bricks | ||
+ | |||
+ | * Digestion to confirm plasmid extraction. | ||
+ | <br> | ||
+ | Methods: | ||
+ | |||
+ | * 3 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_R0010 <span style="color: black;">placI</span>] or [http://partsregistry.org/Part:BBa_R0080 <span style="color: black;">pAraC</span>] bricks on [http://partsregistry.org/Part:pSB1A2 <span style="color: black;">pSB1A2</span>] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our [https://2009.igem.org/Wiki/Team:Warsaw/protocols <span style="color: black;">standard procedure</span>]. The pellet from 5 ml of bacteria was used. | ||
+ | * DNA that should contain [http://partsregistry.org/Part:BBa_R0080 <span style="color: black;">pAraC</span>] on [http://partsregistry.org/Part:pSB1A2 <span style="color: black;">pSB1A2</span>] plasmid was digested with BamHI and PvuI, [http://partsregistry.org/Part:BBa_R0010 <span style="color: black;">placI</span>] on [http://partsregistry.org/Part:pSB1A2 <span style="color: black;">pSB1A2</span>] 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. | ||
+ | <br> | ||
{| class="wikitable" style="text-align:center; width:500px; height:90px; border-collapse: collapse; border: 1px dashed blue; margin: 1em auto 1em auto" border="1" | {| class="wikitable" style="text-align:center; width:500px; height:90px; border-collapse: collapse; border: 1px dashed blue; margin: 1em auto 1em auto" border="1" | ||
|- | |- | ||
! DNA sample | ! DNA sample | ||
! restriction enzymes | ! restriction enzymes | ||
- | ! expected fragments | + | ! expected fragments [bp] |
|- | |- | ||
- | | | + | | [http://partsregistry.org/Part:BBa_R0010 <span style="color: black;">placI on pSB1A2</span>] |
- | | PvuI PvuII | + | | PvuI, PvuII |
- | | 728, | + | | 728, 1551 |
|- | |- | ||
- | | | + | | [http://partsregistry.org/Part:BBa_R0080 <span style="color: black;">pAraC on pSB1A2</span>] |
- | | BamHI PvuI | + | | BamHI, PvuI |
| 782, 1446 | | 782, 1446 | ||
|} | |} | ||
+ | <br> | ||
+ | <br> | ||
+ | Results: | ||
+ | |||
+ | [[image:110709_trawienia_pAraC_i_placI_Franek.JPG]] | ||
+ | <br> | ||
+ | * obtained fragments match expected | ||
+ | <!-- TU PISZ CO CHCESZ! --> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | ===<div style="text-align: center;">Making of RBS-cI part</div>=== | ||
+ | |||
+ | '''Jarek''' | ||
+ | |||
+ | Tasks: | ||
+ | |||
+ | * Transformation of ligation from previous day into competent E.coli strain Top10 | ||
+ | |||
+ | * Plating of transformated culture on the LB+Amp plates and incubation in 37C degree. | ||
+ | <br> | ||
+ | ===Evaluating the quality of bacterial medium=== | ||
+ | |||
+ | '''Andrzej''' | ||
+ | __NOEDITSECTION__ | ||
+ | Tasks: | ||
+ | *Transformation of ''E. coli'' strain DH5alpha with pKs vector as a positive control | ||
+ | Methods: | ||
+ | *If you want to see detailed procedure go [https://2009.igem.org/Team:Warsaw/Calendar-Main/7_July_2009 here] | ||
+ | *Bacteria was plated on the medium containing kanamycin | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
- | |||
+ | ===Franek lub Monika opisze minilizy dla Moniki === | ||
+ | __NOEDITSECTION__ | ||
<!-- do not remove this! --> | <!-- do not remove this! --> | ||
{{WarNotebookEnd}} | {{WarNotebookEnd}} |
Latest revision as of 20:26, 6 September 2009
11 July 2009
Insertion of the pho gene into the pKSII+ plasmid
Kama
Tasks:
- 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].
- purified inserts of biggest concentration were taken to next steps of cloning (marked with an arrow, ~30ng/μl)
Cloning of 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:
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Transformation of chemocompetent strain of E. with [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 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:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0051 BBa_R0051] - promoter (lambda cI regulated); [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 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 |
- obtained fragments match expected
Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Tasks:
- Alkaline lysis of bacterial cultures to obtain plasmids containing [http://partsregistry.org/Part:BBa_R0010 BBa_R0010 - lacI regulated promoter] and [http://partsregistry.org/Part:BBa_R0080 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 [http://partsregistry.org/Part:BBa_R0010 placI] or [http://partsregistry.org/Part:BBa_R0080 pAraC] bricks on [http://partsregistry.org/Part:pSB1A2 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 [http://partsregistry.org/Part:BBa_R0080 pAraC] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid was digested with BamHI and PvuI, [http://partsregistry.org/Part:BBa_R0010 placI] on [http://partsregistry.org/Part:pSB1A2 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] |
---|---|---|
[http://partsregistry.org/Part:BBa_R0010 placI on pSB1A2] | PvuI, PvuII | 728, 1551 |
[http://partsregistry.org/Part:BBa_R0080 pAraC on pSB1A2] | BamHI, PvuI | 782, 1446 |
Results:
- obtained fragments match expected
Making of RBS-cI part
Jarek
Tasks:
- Transformation of ligation from previous day into competent E.coli strain Top10
- Plating of transformated culture on the LB+Amp plates and incubation in 37C degree.
Evaluating the quality of bacterial medium
Andrzej
Tasks:
- Transformation of E. coli strain DH5alpha with pKs vector as a positive control
Methods:
- If you want to see detailed procedure go here
- Bacteria was plated on the medium containing kanamycin
Franek lub Monika opisze minilizy dla Moniki
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