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From 2010.igem.org

Week7 Highlights

Half of our team received BIF confocal microscopy training. We determined that our Amp plates were bad because of excess growth occurring on our overnight transformation plates. We began PCRing our pMAL vector. We continued having problems with 3A assembly. We tried using 1ug of starting DNA instead of 500ng. We also tried following Knight's 3A assembly protocol. All of our assemblies still failed.

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7/26/10

BIF confocal microscopy training

Kit to stock of 1-6G and 1-12O

Ran a gel of the pMAL PCR products --> very faint bands

Used pMAL PCR products as template for another PCR reaction

Ligated LacP1-GFP and transformed using our own TOP10 competent cells

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7/27/10

Excessive growth of 1-6G and 1-12O plates --> amp plates are possibly bad

Gel extraction LacP1, GFP, Linear Tet backbone plasmid restriction digest products

Ran a gel of digestion and ligation products; conclusion: linear plasmid PCR products are bad. Also threw out other assortment of degraded DNA.

3A Assembly using 1 ug of starting DNA.

Poured new Tet and Chl plates

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7/28/10

Followed Knight's 3A Assembly for LacP1-GFP part --> No growth

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7/29/10

3A Assembly using Knight protocol restriction digests --> but replaced linear backbone plasmid with circular plasmid miniprepped from IGEM kit

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7/30/10

LACP1 with GFP in CP-C yielded good results

Ligations (CP1, LACP1, LACPI1 with GFP1 in CP-C)

Redid PCR of CHS3 for pMAL insert

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