• 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 Micropipettes1) Check the volume2) Attach disposable tip (different tips for each size micropipette)3) Depress the plunger (before you put in sample) to the first stop4) Immerse tip in sample5) Draw up the sample slowly and evenly, NOT all at once6) Pause7) Withdraw the tip8) Dispense the sample along the inside wall of tube to help coax out the

sample9) Slowly depress plunger to second stop to expel last bit of fluid, and hold

plunger down in the position10) While holding plunger DOWN withdraw the pipette11) Release plunger12) 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