Team:Minnesota/Notebook

From 2009.igem.org

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<li>Purify the PCR product using [http://2009.igem.org/wiki/images/3/39/DNA_Purification.pdf|QIAquick PCR purification]</li>
<li>Purify the PCR product using [http://2009.igem.org/wiki/images/3/39/DNA_Purification.pdf|QIAquick PCR purification]</li>
<li>Ligate[http://2009.igem.org/wiki/images/3/39/Ligation_Reaction.pdf|Ligation] the insert into pGLOTopo</li>
<li>Ligate[http://2009.igem.org/wiki/images/3/39/Ligation_Reaction.pdf|Ligation] the insert into pGLOTopo</li>
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<li>The plasmid is [http://2009.igem.org/wiki/images/3/39/Transformation_of_Chemically_Competent_Cells.pdf|transformed] into TOP10 cells</li>
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<li>The plasmid is [http://2009.igem.org/wiki/images/3/39/Transformation_of_Chemically_Competent_Cells.pdf| transformed] into TOP10 cells</li>
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<li>The transformants are plated then screened[http://2009.igem.org/wiki/images/3/39/Screening.pdf|screened] using a florescent camera</li>
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<li>The transformants are plated then screened [http://2009.igem.org/wiki/images/3/39/Screening.pdf| screened] using a florescent camera</li>
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<li>Once the colonies have been screened, positive colonies have their plasmids isolated[http://2009.igem.org/wiki/images/3/39/Plasmid_Prep_from_Cultures.pdf|plasmids isolated].</li>
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<li>Once the colonies have been screened, positive colonies have their plasmids isolated[http://2009.igem.org/wiki/images/3/39/Plasmid_Prep_from_Cultures.pdf| plasmids isolated].</li>
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<li>The plasmids are then [http://2009.igem.org/wiki/images/3/39/Sequencing.pdf|sequenced] to ensure the correct sequence was obtained.</li>
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<li>The plasmids are then [http://2009.igem.org/wiki/images/3/39/Sequencing.pdf| sequenced] to ensure the correct sequence was obtained.</li>
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<li></li>
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<li>Transform the cells into DH5αPro and TOP10 and perform [http://2009.igem.org/wiki/images/3/39/Sample_Collection.pdf| sample collection]</li>
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<li>The samples are left at 4 °C for 24 hours.</li>
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<li>The relative GFP intensity is taken with FACSCalibar flow cytometer.</li>
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</ol>
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Revision as of 19:23, 9 August 2009

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Home The Team The Project SynBioSS Designer Modeling Experimental Competition Requirements

Contents

Standard Protocols We Used in the Wet Lab

These link to pdf files:

Bacteria Culture Protocols

Transformation of Chemically Competent Cells

Plasmid Prep from Cultures

DNA Quantification

Polymerase Chain Reaction (PCR)

Restriction Digest

Vector Dephosphorylation

DNA Fragment Ligation

DNA Purification

Sequencing

Preparing Competent Cells

SOEing PCR

Ligation

Screening

Sample Collection

Procedure

  1. Prepare Competent Cells for TOP10 and DH5αPro
  2. Design the primers so that the proper mutations exist in the palindromic sequence of the Tet operator site. Design for each construct: TNN, TTN, and TTL.
  3. Combine Soeing PCR reagents with primers and place in a thermocycler.
  4. Amplify the insert using PCR
  5. Purify the PCR product using PCR purification
  6. Ligate[1] the insert into pGLOTopo
  7. The plasmid is transformed into TOP10 cells
  8. The transformants are plated then screened screened using a florescent camera
  9. Once the colonies have been screened, positive colonies have their plasmids isolatedplasmids isolated.
  10. The plasmids are then sequenced to ensure the correct sequence was obtained.
  11. Transform the cells into DH5αPro and TOP10 and perform sample collection
  12. The samples are left at 4 °C for 24 hours.
  13. The relative GFP intensity is taken with FACSCalibar flow cytometer.


Our Google Calendar

This calendar contains a day-by-day catalog of what we did in the wet lab for our project and parts characterization. The calendar for computational work can be found below and on the Modeling page. Please click on each event to see a detailed description of what we did.



Notebook




June
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30
July
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31
August
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
September
MTWTFSS
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
October
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31