Transformation is the process of introducing DNA into bacterial cells.

Plasmid Plasmids are self- replicating, gene-containing circular pieces of DNA about 1%-5% the size of the bacterial chromosome. Plasmids contain three important parts: – Ori (origin) – Restriction Enzymes: all the different letters and numbers – Antibiotic resistance genes The antibiotic resistance genes are important for our experiment.

Transformation and Antibiotic Resistance Bacteria that have taken up plasmids by transformation will be resistant to certain antibiotics. In today’s experiment we are taking a plasmid with a resistance gene to Kanamycin. We will then introduce this plasmid into the bacterium which will make the bacteria resistant to Kanamycin. If we make agar plates with Kanamycin in them do you think the bacteria will grow? How about Ampicillin (another antibiotic)?

You will perform 2 transformations and have 2 untransformed controls. – Untransformed Bacteria: LB agar plate + Kanamycin LB agar plate + Ampicillin – Transformed Bacteria with pEGFP-C1 LB agar plate + Kanamycin LB agar plate + Ampicillin

Centrifuge Centrifuge: we use this device to place the heavier substances at the bottom of the tubes and lighter at top for easier removal. Balance: When using the centrifuge we have to balance our tubes for equilateral rotation of machine. For example there can NOT be only one tube in the centrifuge.

Micropipettes

Step-by-Step to use Micropipettes 1) Check the volume 2) Attach disposable tip (different tips for each size micropipette) 3) Depress the plunger (before you put in sample) to the first stop 4) Immerse tip in sample 5) Draw up the sample slowly and evenly, NOT all at once 6) Pause 7) Withdraw the tip 8) Dispense the sample along the inside wall of tube to help coax out the sample 9) Slowly depress plunger to second stop to expel last bit of fluid, and hold plunger down in the position 10) While holding plunger DOWN withdraw the pipette 11) Release plunger 12) Discard the tip *Always Change tips for each new reagent you need to pipette or if you touch any other liquids. We want to remember our ASEPTIC techniques!

300µl (p1000 pipette)

120µl (p200 pipette)

50µl (p200 pipette)

Glass Rods