Team:Montreal/Notebook

From 2008.igem.org

Revision as of 03:14, 17 June 2008

Lab Protocols

1. Making a DNA Agarose Gel

2. Cell Transformation

3. Midiprep with Eppendorf Tubes

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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