Team:Montreal/Notebook

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==Lab Protocols==
==Lab Protocols==
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Key Elements of the Central Dogma of cloning
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1. [https://2008.igem.org/Team:Montreal/Restriction_digest Restriction Digest]
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1. [https://2008.igem.org/Team:Montreal/DNA_Agarose_Gel Making a DNA Agarose Gel]
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2. [https://2008.igem.org/Team:Montreal/DNA_Agarose_Gel Making a DNA Agarose Gel]
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2. [https://2008.igem.org/Team:Montreal/Cell_transformation Cell Transformation]
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3. [https://2008.igem.org/Team:Montreal/Gel_extraction Gel extraction]
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3. [https://2008.igem.org/Team:Montreal/Midiprep Midiprep with Eppendorf Tubes]
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4. [https://2008.igem.org/Team:Montreal/Confirmation_quantity DNA quantity assessment]
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5. [https://2008.igem.org/Team:Montreal/Ligation Ligation]
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6. [https://2008.igem.org/Team:Montreal/Cell_transformation Cell Transformation]
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7. [https://2008.igem.org/Team:Montreal/Seeding Seeding method]
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8. [https://2008.igem.org/Team:Montreal/Midiprep DNA Extraction: Mini-, Midi-, Maxiprep]
==Lab Progress==
==Lab Progress==

Revision as of 03:19, 17 June 2008

Home The Team The Project Parts Submitted to the Registry Modeling Notebook
Central dogma.jpg

Lab Protocols

Key Elements of the Central Dogma of cloning 1. Restriction Digest

2. Making a DNA Agarose Gel

3. Gel extraction

4. DNA quantity assessment

5. Ligation

6. Cell Transformation

7. Seeding method

8. DNA Extraction: Mini-, Midi-, Maxiprep

Lab Progress

May 21st, 2008:

    Restriction enzyme digest was done on the J-brick with EcoRI. Gel was run on the J-brick after the restriction digest. No DNA was detected on the gel (the ladder was visible on the gel).

May 22nd, 2008:

    Prepared TOP10 competent cells for eventual transformation.
    Performed Mini-prep on Reporter+ Cells
    Performed Digest and Gel on Reporter plasmid extract -- no DNA present, suggest follow-up maxi-prep

May 23rd, 2008:

    Transformed Top10 cells with Puc19 to ensure that the competent cell procedure was successful. Growth was observed, therefore procedure was successful.

May 25th, 2008:

    Diluted Reporter cells 1/1000 with 5ul Kan/mL culture for 16h incubation at 5:30pm. To be used for Maxiprep at 9:30am-1:30pm.

May 28th, 2008:

    Ran gel on Elowitz Reporter DNA cut with EcoR1; 2 bands
    0.7 kb and 2.0 kb, confirms identity of reporter DNA.
    Seeded syn-I and J-40001 into amp/kan LB and kan LB.

May 29th, 2008:

    Growth of J-brick in culture - No growth of I-brick on culture
    Seeded J-brick for Midi-Prep in 40mL LB with ampicillin
    Transformed TOP10 cells with both I brick and Reporter Plasmid

June 2nd, 2008:

    Growth of I-brick on culture
    Midi-prep of both I and J brick followed by gel
    Gel indicates no presence of DNA, will be confirmed by spectrophotometric assay

June 3rd, 2008:

    Seeding of 5mL cultures of both I and J brick
    Identity of colonies on I brick plates is suspect, must ensure that eventual DNA gel confirms exact restriction digest

June 4th, 2008:

    Midiprep was done on the I and the J-brick. Once the isopropanol was added, the J-brick midiprep looked clear (no DNA was eluted). DNA gel needs to be done to confirm presence of DNA in both cases.

June 9th, 2008:

    Seeded J and I-brick re-seeded for Maxi-Prep
    Gel failed to confirm presence of previously collected DNA samples of J and I-brick, will be repeated following Maxi-Prep.

June 10th, 2008:

    Since no growth was observed in the I-brick culture, the I brick was re-seeded.
    The J-brick was diluted in 500-ml of LB broth(for a Maxiprep to be done the next day).

June 11th, 2008:

    Maxiprep of the J-brick.
    Restriction digest of J-brick.
    Dilution of I-brick in 500-ml of LB broth.

June 12th, 2008:

    Maxiprep of the I-brick.
    Restriction digest and gel of June 11th and June 12th I-brick and J-brick DNA using EcoR1. Bands revealed at roughly 4000bp and 2500bp for I-brick (expected 2652bp and 3939bp). J brick single band that was not informative, new digestion to be completed tomorrow.

June 16th, 2008:

    I-brick was seeded and diluted over last two days, but there was insufficient growth so it will be left to grow one more day before performing another midi-prep. This is to compliment the already successful Maxi-Prep that gave low concentrations of DNA.
    Another gel was performed of previous J-brick preps that confirmed the absence of the desired plasmid, no DNA was detected when digested with EcoR1.
    J-brick was re-transformed into TOP10 chemically competent cells and then plated on Amp+ plates.<ul/></p>

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