Team:University of Lethbridge/Notebook/GeneralLabSeptember

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Nathan Puhl, Roxanne
-Transformed DH5a cells with the biobrick part R0011 (pLacI) obtained from the 2007 iGEM parts. Plated on LB + amp agar plates.

Roxanne
-Picked 3 representative colonies and incubated them in LB+amp media overnight at 37.0C.

Roxanne
-plasmid prepped the pLacI cells which had been incubated the night before. Ran plasmids on a 1% Agarose Gel at 100V for 27 minutes

Nathan Puhl
-Visualized the Gel and Glycerol Stocked the Cells.

Roxanne, Nathan Puhl
-Performed a Restriction Digest of I13504 (RBS+GFP+dT) and R0011 (pLacI recombinant)

-10 uL template DNA -0.5 uL Restriction Enzyme #1 -0.5 uL Restriction Enzyme #2 -2 uL NEB 2 -7 uL ddH2O _________ 20 uL Reaction overnight at 37.0C

Roxanne, Nathan Puhl
-Have been working on Cloning the Reporter System. Transformation worked, however, the lack of Red colonies on the RFP plates and sub-culture have led us to believe that something didn't work somewhere along the way.

Roxanne
-Streaked Plates of RFP sub, TetR sub and pLacI

Roxanne
-Picked colonies from the RFP sub, TetR sub and pLacI plates. Incubating overnight at 37.0C in LB media + amp.

Nathan Puhl
-Plasmid prepped the RFP sub, TetR sub and pLacI cultures.

Nathan Puhl, Roxanne
-Restriction digest of the RFP sub and TetR sub with XbaI and PstI -Restriction Digest of pLacI with SpeI and PstI.

Nathan Puhl
-Ran a 1% agarose gel of the digestion products. RFP and TetR look good, however, pLacI is running high.

Nathan Puhl, Roxanne
-repicked colonies from pLacI plates for subculture in LB media.

Nathan Puhl, Roxanne
-plasmid prepped the pLacI culture. -Ran plasmids on a 1% agarose gel. -Oops, sub-culture it again.

Roxanne
-plasmid prepped the pLacI culture. -Ran the gel of the plasmid prep products



Roxanne
-Gel Extraction of pLacI x2, RFP sub, and TetR sub



-Ran a gel of the extraction products, however, no product appears.

Roxanne
-Ran the gel of the extraction products again. -1 uL of DNA on a 1% agarose gel @ 100V for 25 minutes, with a 1kb ladder. -The Kit ate my DNA again!! -Repicked colonies from the plate and incubated in LB+amp media overnight @ 37.0C

Roxanne
-Took the tubes out of the shaker and put in fridge to be plasmid prepped at a later date.

Roxanne
-plasmid prepped the pLacI, RFP and TetR genes -Ran the plasmids on a gel. RFP and TetR look good, pLacI is a little wonky, not comforteble with it -Setup a Restriction Digest of RFP and TetR, using XbaI and PstI. -Repicked te pLacI colony and incubated in LB+amp overnight @ 37.0C

Roxanne
-Plasmid prepped the pLacI gene and put te plasmid in the freezer -Inactivated the enzymes from the RFP and TetR double digest

Roxanne
-Testing the Enzymes we received from Fermentas which were sent without ice on the pLacI gene -Cut Half of my plasmid prep with the iGEM SpeI and PstI, and the other half with H-J's PstI and the iGEM SpeI.